AU2014274643B2 - Recombinant bacteria for producing glycerol and glycerol-derived products from sucrose - Google Patents

Abstract

Recombinant bacteria capable of producing glycerol and glycerol derived products from sucrose are described. The recombinant bacteria comprise in their genome or on at least one recombinant construct: a 5 nucleotide sequence encoding a polypeptide having sucrose transporter activity; a nucleotide sequence encoding a polypeptide having fructokinase activity; and a nucleotide sequence encoding a polypeptide having sucrose hydrolase activity. These nucleotide sequences are each operably linked to the same or a different promoter. These io recombinant bacteria are capable of metabolizing sucrose to produce glycerol and/or glycerol-derived products such as 1,3-propanediol and 3 hydroxypropionic acid.

Description

P/00/01I Regulation 3.2 AUSTRALIA Patents Act 1990 COMPLETE SPECIFICATION FOR A DIVISIONAL PATENT ORIGINAL Name of Applicant: E. I. DU PONT DE NEMOURS AND COMPANY Actual Inventors: Andrew C. ELIOT Anthony A. GATENBY Tina K. VAN DYK Address for Service: Houlihan 2 , Level 1, 70 Doncaster Road, Balwyn North, Victoria 3104, Australia Invention Title: RECOMBINANT BACTERIA FOR PRODUCING GLYCEROL AND GLYCEROL-DERIVED PRODUCTS FROM SUCROSE The following statement is a full description of this invention, including the best method of performing it known to the Applicant: 1 TITLE RECOMBINANT BACTERIA FOR PRODUCING GLYCEROL AND GLYCEROL-DERIVED PRODUCTS FROM SUCROSE 5 The present application is a divisional application from Australian patent application number 2010325895. The entire disclosures of Australian patent application number 2010325895 and its corresponding International application, PCT/US2010/058832, are incorporated herein by reference. 10 FIELD OF THE INVENTION The invention relates to the fields of microbiology and molecular biology. More specifically, recombinant bacteria having the ability to produce glycerol and glycerol-derived products using sucrose as a carbon 15 source and methods of utilizing such recombinant bacteria are provided. BACKGROUND OF THE INVENTION Many commercially useful microorganisms use glucose as their main carbohydrate source. However, a disadvantage of the use of 20 glucose by microorganisms developed for production of commercially desirable products is the high cost of glucose. The use of sucrose and mixed feedstocks containing sucrose and other sugars as carbohydrate sources for microbial production systems would be more commercially desirable because these materials are readily available at a lower cost. 25 A production microorganism can function more efficiently when it can utilize any sucrose present in a mixed feedstock. Therefore, when a production microorganism does not have the ability to utilize sucrose efficiently as a major carbon source, it cannot operate as efficiently. For example, bacterial cells typically show preferential sugar use, with 30 glucose being the most preferred. In artificial media containing mixtures of sugars, glucose is typically metabolized to its entirety ahead of other sugars. Moreover, many bacteria lack the ability to utilize sucrose. For 2 example, less than 50% of Escherichia coli strains have the ability to utilize sucrose. Thus, when a production microorganism cannot utilize sucrose as a carbohydrate source, it is desirable to engineer the microorganism so that it can utilize sucrose. 5 Recombinant bacteria that have been engineered to utilize sucrose by incorporation of sucrose utilization genes have been reported. For example, Livshits et al. (U.S. Patent No. 6,960,455) describe the production of amino acids using Escherichia coli strains containing genes encoding a metabolic pathway for sucrose utilization. Additionally, Olson 10 et al. (Appl. MicrobioL. Biotechnol. 74:1031-1040, 2007) describe Escherichia coli strains carrying genes responsible for sucrose degradation, which produce L-tyrosine or L-phenylalanine using sucrose as a carbon source. However, there is a need for bacterial strains that are capable of producing glycerol and glycerol-derived products using 15 sucrose as carbon source. SUMMARY OF THE INVENTION In one embodiment, the invention provides a recombinant bacterium comprising in its genome or on at least one recombinant 20 construct: (a) one or more nucleotide sequences encoding a polypeptide or a polypeptide complex having sucrose transporter activity; (b) a nucleotide sequence encoding a polypeptide having fructokinase activity; and 25 (c) a nucleotide sequence encoding a polypeptide having sucrose hydrolase activity; wherein (a), (b) and (c) are each operably linked to the same or a different promoter, further wherein said recombinant bacterium is capable of metabolizing sucrose to produce a product selected from the group 30 consisting of glycerol, 1,3-propanediol and 3-hydroxypropionic acid. In a second embodiment, the invention provides a process for making glycerol, 1,3-propanediol and/or 3-hydroxypropionic acid from 3 sucrose comprising: a) culturing the recombinant bacterium disclosed herein in the presence of sucrose; and b) optionally, recovering the glycerol, 1,3-propanediol and/or 3 5 hydroxypropionic acid produced. BRIEF SEQUENCE DESCRIPTIONS The following sequences conform with 37 C.F.R. 1.821 1.825 ("Requirements for Patent Applications Containing Nucleotide Sequences io and/or Amino Acid Sequence Disclosures - the Sequence Rules") and consistent with World Intellectual Property Organization (WIPO) Standard ST.25 (1998) and the sequence listing requirements of the EPO and PCT (Rules 5.2 and 49.5(a bis), and Section 208 and Annex C of the Administrative Instructions). The symbols and format used for nucleotide 15 and amino acid sequence data comply with the rules set forth in 37 C.F.R. §1.822. 4 Table A Summary of Gene and Protein SEQ ID Numbers Gene Coding Encoded Sequence Protein SEQ ID NO: SEQ ID NO: GPD1 from Saccharomyces cerevisiae 1 2 GPD2 from Saccharomyces cerevisiae 3 4 GPP1 from Saccharomyces cerevisiae 5 6 GPP2 from Saccharomyces cerevisiae 7 8 dhaB1 from Klebsiella pneumoniae 9 10 dhaB2 from Klebsiella pneumoniae 11 12 dhaB3 from Klebsiella pneumoniae 13 14 aldB from Escherichia coli 15 16 aldA from Escherichia coli 17 18 aldH from Escherichia coli 19 20 galP from Escherichia coli 21 22 cscB from Escherichia coli EC3132 23 24 cscB from Escherichia coli ATCC13281 25 26 cscB from Bifidobacterium lactis 27 28 susT1 from Streptococcus pneumoniae 29 30 strain TIGR4 susT2 from Streptococcus pneumoniae 31 32 strain TIGR4 susX from Streptococcus pneumoniae 33 34 strain TIGR4 malE from Streptococcus mutans 35 36 malF from Streptococcus mutans 37 38 maIG from Streptococcus mutans 39 40 malK from Streptococcus mutans 41 42 scrK from Agrobacterium tumefaciens 43 44 scrK from Streptococcus mutans 45 46 scrK From Escherichia coli 84 85 scrK from Klebsiella pneumoniae 86 87 cscK from Escherichia coli 47 48 cscK from Enterococcus faecalis 49 50 HXK1 from Saccharomyces cerevisiae 51 52 HXK2 from Saccharomyces cerevisiae 53 54 cscA from Escherichia coli EC3132 55 56 cscA from Escherichia coli ATCC13281 57 58 bfrA from Bifidobacterium lactis strain DSM 59 60 10140T SUC2 from Saccharomyces cerevisiae 61 62 scrB from Corynebacterium glutamicum 63 64 sucrose phosphorylase gene from 65 66 Leuconostoc mesenteroides DSM 20193 sucP Bifidobacterium adolescentis DSM 67 68 20083 dhaTfrom Klebsiella pneumoniae 69 70 5 SEQ ID NO:71 is the nucleotide sequence of the coding region of the dhaX gene from Klebsiella pneumoniae. SEQ ID NO:72 is the nucleotide sequence of plasmid pSYCO1 01. SEQ ID NO:73 is the nucleotide sequence of plasmid pSYCO1 03. 5 SEQ ID NO:74 is the nucleotide sequence of plasmid pSYCO1 06. SEQ ID NO:75 is the nucleotide sequence of plasmid pSYCO1 09. SEQ ID NO:76 is the nucleotide sequence of plasmid pSYCO400/AGRO. SEQ ID NO:77 is the nucleotide sequence of plasmid pScrl io described in Example 1 herein. SEQ ID NO:78 is the nucleotide sequence of plasmid pBHRcscBKA described in Example 1 herein. SEQ ID NO:79 is the nucleotide sequence of plasmid pBHRcscBKAmutB described in Example 1 herein. 15 SEQ ID NOs:80-83 are the nucleotide sequences of primers used to construct strain TTab described in Examples 2-4 herein. DETAILED DESCRIPTION The disclosure of each reference set forth herein is hereby 20 incorporated by reference in its entirety. As used herein and in the appended claims, the singular forms "a", "an", and "the" include plural reference unless the context clearly dictates otherwise. Thus, for example, reference to "a cell" includes one or more cells and equivalents thereof known to those skilled in the art, and so forth. 25 In the context of this disclosure, a number of terms and abbreviations are used. The following definitions are provided. "Open reading frame" is abbreviated as "ORF". "Polymerase chain reaction" is abbreviated as "PCR". "American Type Culture Collection" is abbreviated as "ATCC". 30 The term "recombinant glycerol-producing bacterium" refers to a bacterium that has been genetically engineered to be capable of producing glycerol and/or glycerol-derived products such as 1,3 6 propanediol and 3-hydroxypropionic acid. The term "polypeptide or polypeptide complex having sucrose transporter activity" refers to a polypeptide or polypeptide complex that is capable of mediating the transport of sucrose into microbial cells. 5 Examples of polypeptides having sucrose transporter activity include, but are not limited to, sucrose:H+ symporters. Examples of polypeptide complexes having sucrose transporter activity include, but are not limited to, ABC-type transporters. Sucrose:H+ symporters are encoded by, for example, the cscB gene found in E. coli strains such as EC3132 (Jahreis 10 et al., J. Bacteriol. 184:5307-5316, 2002) or ATCC1 3281 (Olson et al., Apple. Microbiol. Biotechnol. 74:1031-1040, 2007), and Bifidobacterium lactis strain DSM 10 14 0 T (Ehrmann et al., Curr. Microbiol. 46(6):391-397, 2003). An example of an ABC-type transporter with activity towards sucrose is the complex encoded by the genes susT1, susT2 and susX in 15 Streptococcus pneumoniae strain TIGR4 (lyer and Camilli, Molecular Microbiology 66:1-13, 2007). Polypeptides or polypeptide complexes having sucrose transporter activity may also have activity towards other saccharides. An example is the maltose transporter complex of Streptococcus mutans encoded by ma/EFGK (Kilic et al., FEMS Microbiol 20 Lett. 266:218, 2007). The term "polypeptide having fructokinase activity" refers to a polypeptide that has the ability to catalyze the conversion of D-fructose + ATP to fructose-phosphate + ADP. Typical of fructokinase is EC 2.7.1.4. Enzymes that have some ability to phosphorylate fructose, 25 whether or not this activity is their predominant activity, may be referred to as a fructokinase. Abbreviations used for genes encoding fructokinases and proteins having fructokinase activity include, for example, "Frk', "scrK', "cscK', "FK', and "KHK'. Fructokinase is encoded by the scrK gene in Agrobacterium tumefaciens and 30 Streptococcus mutans; and by the cscK gene in certain Escherichia coli strains. The term "polypeptide having sucrose hydrolase activity" refers to 7 a polypeptide that has the ability to catalyze the hydrolysis of sucrose to produce glucose and fructose. Such polypeptides are often referred to as "invertases" or "p-fructofuranosidases". Typical of these enzymes is EC 3.2.1.26. Examples of genes encoding polypeptides having sucrose 5 hydrolase activity are the cscA gene found in E. coli strains EC3132 (Jahreis et al. supra) or ATCC1 3281 (Olson et al., supra), the bfrA gene from Bifidobacterium lactis strain DSM 10 1 4 0 T, and the SUC2 gene from Saccharomyces cerevisiae (Carlson and Botstein, Cell 28:145, 1982). A polypeptide having sucrose hydrolase activity may also have sucrose 10 phosphate hydrolase activity. An example of such a peptide is encoded by scrB in Corynebacterium glutamicum (Engels et al., FEMS Microbiol Lett. 289:80-89, 2008). A polypeptide having sucrose hydrolase activity may also have sucrose phosphorylase activity. Typical of such an enzyme is EC 2.4.1.7. Examples of genes encoding sucrose 15 phosphorylases having sucrose hydrolase activity are found in Leuconostoc mesenteroides DSM 20193 (Goedl et al., Journal of Biotechnology 129:77-86, 2007) and Bifidobacterium adolescents DSM 20083 (van den Broek et al., Appl. Microbio/. Biotechnol. 65:219-227, 2004), among others. 20 The terms "glycerol derivative" and "glycerol-derived products" are used interchangeably herein and refer to a compound that is synthesized from glycerol or in a pathway that includes glycerol. Examples of such products include 3-hydroxypropionic acid, methylglyoxal, 1,2-propanediol, and 1,3-propanediol. 25 The term "microbial product" refers to a product that is microbially produced, i.e., the result of a microorganism metabolizing a substance. The product may be naturally produced by the microorganism, or the microorganism may be genetically engineered to produce the product. The terms "phosphoenolpyruvate-sugar phosphotransferase 30 system", "PTS system", and "PTS" are used interchangeably herein and refer to the phosphoenolpyruvate-dependent sugar uptake system. The terms "phosphocarrier protein HPr" and "PtsH" refer to the 8 phosphocarrier protein encoded by ptsH in E. coli. The terms "phosphoenolpyruvate-protein phosphotransferase" and "Ptsl" refer to the phosphotransferase, EC 2.7.3.9, encoded by ptsl in E. coli. The terms "glucose-specific IIA component", and "Crr" refer to enzymes 5 designated as EC 2.7.1.69, encoded by crr in E. coli. PtsH, Ptsl, and Crr comprise the PTS system. The term "PTS minus" refers to a microorganism that does not contain a PTS system in its native state or a microorganism in which the PTS system has been inactivated through the inactivation of a PTS gene. 10 The terms "glycerol-3-phosphate dehydrogenase" and "G3PDH" refer to a polypeptide responsible for an enzyme activity that catalyzes the conversion of dihydroxyacetone phosphate (DHAP) to glycerol 3 phosphate (G3P). In vivo G3PDH may be NAD- or NADP-dependent. When specifically referring to a cofactor specific glycerol-3-phosphate 15 dehydrogenase, the terms "NAD-dependent glycerol-3-phosphate dehydrogenase" and "NADP-dependent glycerol-3-phosphate dehydrogenase" will be used. As it is generally the case that NAD dependent and NADP-dependent glycerol-3-phosphate dehydrogenases are able to use NAD and NADP interchangeably (for example by the 20 enzyme encoded by gpsA), the terms NAD-dependent and NADP dependent glycerol-3-phosphate dehydrogenase will be used interchangeably. The NAD-dependent enzyme (EC 1.1.1.8) is encoded, for example, by several genes including GPD1, also referred to herein as DAR1 (coding sequence set forth in SEQ ID NO:1; encoded protein 25 sequence set forth in SEQ ID NO:2), or GPD2 (coding sequence set forth in SEQ ID NO:3; encoded protein sequence set forth in SEQ ID NO:4), or GPD3. The NADP-dependent enzyme (EC 1.1.1.94) is encoded, for example, by gpsA. The terms "glycerol 3-phosphatase", "sn-glycerol 3-phosphatase", 30 "D,L-glycerol phosphatase", and "G3P phosphatase" refer to a polypeptide having an enzymatic activity that is capable of catalyzing the conversion of glycerol 3-phosphate and water to glycerol and inorganic phosphate. G3P 9 phosphatase is encoded, for example, by GPP1 (coding sequence set forth in SEQ ID NO:5; encoded protein sequence set forth in SEQ ID NO:6), or GPP2 (coding sequence set forth in SEQ ID NO:7; encoded protein sequence set forth in SEQ ID NO:8). 5 The term "glycerol dehydratase" or "dehydratase enzyme" refers to a polypeptide having enzyme activity that is capable of catalyzing the conversion of a glycerol molecule to the product, 3-hydroxypropionaldehyde (3-HPA). For the purposes of the present invention the dehydratase enzymes 10 include a glycerol dehydratase (E.C. 4.2.1.30) and a diol dehydratase (E.C. 4.2.1.28) having preferred substrates of glycerol and 1,2-propanediol, respectively. Genes for dehydratase enzymes have been identified in Klebsiella pneumoniae, Citrobacter freundii, Clostridium pasteurianum, Salmonella typhimurium, Klebsiella oxytoca, and 15 Lactobacillus reuteri, among others. In each case, the dehydratase is composed of three subunits: the large or "a" subunit, the medium or "$" subunit, and the small or "y" subunit. The genes are also described in, for example, Daniel et al. (FEMS Microbiol. Rev. 22, 553 (1999)) and Toraya and Mori (J. Biol. Chem. 274, 3372 (1999)). Genes encoding the large or 20 "a" (alpha) subunit of glycerol dehydratase include dhaB1 (coding sequence set forth in SEQ ID NO:9, encoded protein sequence set forth in SEQ ID NO:10), gidA and dhaB; genes encoding the medium or "$" (beta) subunit include dhaB2 (coding sequence set forth in SEQ ID NO:1 1, encoded protein sequence set forth in SEQ ID NO:12), gldB, and dhaC; 25 genes encoding the small or "y" (gamma) subunit include dhaB3 (coding sequence set forth in SEQ ID NO:1 3, encoded protein sequence set forth in SEQ ID NO:14), gldC, and dhaE. Other genes encoding the large or "a" subunit of diol dehydratase include pduC and pddA; other genes encoding the medium or "P" subunit include pduD and pddB; and other 30 genes encoding the small or "y" subunit include pduE and pddC. 10 Glycerol and diol dehydratases are subject to mechanism-based suicide inactivation by glycerol and some other substrates (Daniel et al., FEMS Microbiol. Rev. 22, 553 (1999)). The term "dehydratase reactivation factor" refers to those proteins responsible for reactivating 5 the dehydratase activity. The terms "dehydratase reactivating activity", "reactivating the dehydratase activity" and "regenerating the dehydratase activity" are used interchangeably and refer to the phenomenon of converting a dehydratase not capable of catalysis of a reaction to one capable of catalysis of a reaction or to the phenomenon 10 of inhibiting the inactivation of a dehydratase or the phenomenon of extending the useful half-life of the dehydratase enzyme in vivo. Two proteins have been identified as being involved as the dehydratase reactivation factor (see, e.g., U.S. Patent No. 6,013,494 and references therein; Daniel et al., supra; Toraya and Mori, J. Biol. Chem. 274, 3372 15 (1999); and Tobimatsu et al., J. Bacteriol. 181, 4110 (1999)). Genes encoding one of the proteins include, for example, orfZ, dhaB4, gdrA, pduG and ddrA. Genes encoding the second of the two proteins include, for example, orfX, orf2b, gdrB, pduH and ddrB. The terms "1,3-propanediol oxidoreductase", "1,3-propanediol 20 dehydrogenase" and "DhaT" are used interchangeably herein and refer to the polypeptide(s) having an enzymatic activity that is capable of catalyzing the interconversion of 3-HPA and 1,3-propanediol provided the gene(s) encoding such activity is found to be physically or transcriptionally linked to a dehydratase enzyme in its natural (i.e., wild 25 type) setting; for example, the gene is found within a dha regulon as is the case with dhaT from Klebsiella pneumoniae. Genes encoding a 1,3-propanediol oxidoreductase include, but are not limited to, dhaT from Klebsiella pneumoniae, Citrobacter freundii, and Clostridium pasteurianum. Each of these genes encode a polypeptide belonging to 30 the family of type I| alcohol dehydrogenases, which exhibits a conserved iron-binding motif, and has a preference for the NAD+/NADH linked interconversion of 3-HPA and 1,3-propanediol (Johnson and Lin, 11 J. Bacteriol. 169, 2050 (1987); Daniel et al., J. BacterioL. 177, 2151 (1995); and Leurs et al., FEMS MicrobioL. Lett. 154, 337 (1997)). Enzymes with similar physical properties have been isolated from Lactobacillus brevis and Lactobacillus buchneri (Veiga da Dunha and 5 Foster, Appl. Environ. Microbiol. 58, 2005 (1992)). The term "dha regulon" refers to a set of associated polynucleotides or open reading frames encoding polypeptides having various biological activities, including but not limited to a dehydratase activity, a reactivation activity, and a 1,3-propanediol oxidoreductase. io Typically a dha regulon comprises the open reading frames dhaR, orfY, dhaT, orfX, orfW, dhaBl, dhaB2, dhaB3, and orfZ as described in U.S. Patent No. 7,371,558. The terms "aldehyde dehydrogenase" and "Aid" refer to a polypeptide that catalyzes the conversion of an aldehyde to a carboxylic 15 acid. Aldehyde dehydrogenases may use a redox cofactor such as NAD, NADP, FAD, or PQQ. Typical of aldehyde dehydrogenases is EC 1.2.1.3 (NAD-dependent); EC 1.2.1.4 (NADP-dependent); EC 1.2.99.3 (PQQ dependent); or EC 1.2.99.7 (FAD-dependent). An example of an NADP dependent aldehyde dehydrogenase is AldB (SEQ ID NO:16), encoded by 20 the E. coli gene aldB (coding sequence set forth in SEQ ID NO:1 5). Examples of NAD-dependent aldehyde dehydrogenases include AldA (SEQ ID NO:18), encoded by the E. coligene aldA (coding sequence set forth in SEQ ID NO:17); and AldH (SEQ ID NO:20), encoded by the E. coli gene aldH (coding sequence set forth in SEQ ID NO:19). 25 The terms "glucokinase" and "GIk" are used interchangeably herein and refer to a protein that catalyzes the conversion of D-glucose + ATP to glucose 6-phosphate + ADP. Typical of glucokinase is EC 2.7.1.2. Glucokinase is encoded by gik in E. coli. The terms "phosphoenolpyruvate carboxylase" and "Ppc" are 30 used interchangeably herein and refer to a protein that catalyzes the conversion of phosphoenolpyruvate + H 2 0 + C02 to phosphate + oxaloacetic acid. Typical of phosphoenolpyruvate carboxylase is EC 12 4.1.1.31. Phosphoenolpyruvate carboxylase is encoded by ppc in E. coli. The terms "glyceraldehyde-3-phosphate dehydrogenase" and "GapA" are used interchangeably herein and refer to a protein having an 5 enzymatic activity capable of catalyzing the conversion of glyceraldehyde 3-phosphate + phosphate + NAD* to 3-phospho-D glyceroyl-phosphate + NADH + H+. Typical of glyceraldehyde-3 phosphate dehydrogenase is EC 1.2.1.12. Glyceraldehyde-3-phosphate dehydrogenase is encoded by gapA in E. coli. 10 The terms "aerobic respiration control protein" and "ArcA" are used interchangeably herein and refer to a global regulatory protein. The aerobic respiration control protein is encoded by arcA in E. coli. The terms "methylglyoxal synthase" and "MgsA" are used interchangeably herein and refer to a protein having an enzymatic activity 15 capable of catalyzing the conversion of dihydroxyacetone phosphate to methylglyoxal + phosphate. Typical of methylglyoxal synthase is EC 4.2.3.3. Methylglyoxal synthase is encoded by mgsA in E. coli. The terms "phosphogluconate dehydratase" and "Edd" are used interchangeably herein and refer to a protein having an enzymatic 20 activity capable of catalyzing the conversion of 6-phospho-gluconate to 2-keto-3-deoxy-6-phospho-gluconate + H 2 0. Typical of phosphogluconate dehydratase is EC 4.2.1.12. Phosphogluconate dehydratase is encoded by eddin E. coli. The term "YciK" refers to a putative enzyme encoded by yciK 25 which is translationally coupled to btuR, the gene encoding Cob(l)alamin adenosyltransferase in E. coli. The term "cob(I)alamin adenosyltransferase" refers to an enzyme capable of transferring a deoxyadenosyl moiety from ATP to the reduced corrinoid. Typical of cob(I)alamin adenosyltransferase is EC 2.5.1.17. 30 Cob(l)alamin adenosyltransferase is encoded by the gene "btuR" in E. coli, "cobA" in Salmonella typhimurium, and "cobO" in Pseudomonas denitrificans. 13 The terms "galactose-proton symporter" and "GaIP" are used interchangeably herein and refer to a protein having an enzymatic activity capable of transporting a sugar and a proton from the periplasm to the cytoplasm. D-glucose is a preferred substrate for GaIP. Galactose-proton 5 symporter is encoded by ga/P in Escherichia coli (coding sequence set forth in SEQ ID NO:21, encoded protein sequence set forth in SEQ ID NO:22). The term "non-specific catalytic activity" refers to the polypeptide(s) having an enzymatic activity capable of catalyzing the io interconversion of 3-HPA and 1,3-propanediol and specifically excludes 1,3-propanediol oxidoreductase(s). Typically these enzymes are alcohol dehydrogenases. Such enzymes may utilize cofactors other than NAD+/NADH, including but not limited to flavins such as FAD or FMN. A gene for a non-specific alcohol dehydrogenase (yqhD) is found, for 15 example, to be endogenously encoded and functionally expressed within E. coli K-12 strains. The terms "1.6 long GI promoter", "1.20 short/long GI Promoter", and "1.5 long GI promoter" refer to polynucleotides or fragments containing a promoter from the Streptomyces lividans glucose isomerase 20 gene as described in U.S. Patent No. 7,132,527. These promoter fragments include a mutation which decreases their activities as compared to the wild type Streptomyces lividans glucose isomerase gene promoter. The terms "function" and "enzyme function" are used interchangeably herein and refer to the catalytic activity of an enzyme in 25 altering the rate at which a specific chemical reaction occurs without itself being consumed by the reaction. It is understood that such an activity may apply to a reaction in equilibrium where the production of either product or substrate may be accomplished under suitable conditions. 30 The terms "polypeptide" and "protein" are used interchangeably herein. The terms "carbon substrate" and "carbon source" are used 14 interchangeably herein and refer to a carbon source capable of being metabolized by the recombinant bacteria disclosed herein and, particularly, carbon sources comprising fructose and glucose. The carbon source may further comprise other monosaccharides; disaccharides, such 5 as sucrose; oligosaccharides; or polysaccharides. The terms "host cell" and "host bacterium" are used interchangeably herein and refer to a bacterium capable of receiving foreign or heterologous genes and capable of expressing those genes to produce an active gene product. 10 The term "production microorganism" as used herein refers to a microorganism, including, but not limited to, those that are recombinant, used to make a specific product such as 1,3-propanediol, glycerol, 3 hydroxypropionic acid, polyunsaturated fatty acids, and the like. As used herein, "nucleic acid" means a polynucleotide and 15 includes a single or double-stranded polymer of deoxyribonucleotide or ribonucleotide bases. Nucleic acids may also include fragments and modified nucleotides. Thus, the terms "polynucleotide", "nucleic acid sequence", "nucleotide sequence" or "nucleic acid fragment" are used interchangeably herein and refer to a polymer of RNA or DNA that is 20 single- or double-stranded, optionally containing synthetic, non-natural or altered nucleotide bases. Nucleotides (usually found in their 5' monophosphate form) are referred to by their single letter designation as follows: "A" for adenylate or deoxyadenylate (for RNA or DNA, respectively), "C" for cytidylate or deoxycytidylate, "G" for guanylate or 25 deoxyguanylate, "U" for uridylate, "T" for deoxythymidylate, "R" for purines (A or G), "Y" for pyrimidines (C or T), "K" for G or T, "H" for A or C or T, "I" for inosine, and "N" for any nucleotide. A polynucleotide may be a polymer of RNA or DNA that is single- or double-stranded, that optionally contains synthetic, non-natural or altered 30 nucleotide bases. A polynucleotide in the form of a polymer of DNA may be comprised of one or more segments of cDNA, genomic DNA, synthetic DNA, or mixtures thereof. 15 "Gene" refers to a nucleic acid fragment that expresses a specific protein, and which may refer to the coding region alone or may include regulatory sequences preceding (5' non-coding sequences) and following (3' non-coding sequences) the coding sequence. "Native 5 gene" refers to a gene as found in nature with its own regulatory sequences. "Chimeric gene" refers to any gene that is not a native gene, comprising regulatory and coding sequences that are not found together in nature. Accordingly, a chimeric gene may comprise regulatory sequences and coding sequences that are derived from io different sources, or regulatory sequences and coding sequences derived from the same source, but arranged in a manner different than that found in nature. "Endogenous gene" refers to a native gene in its natural location in the genome of an organism. A "foreign" gene refers to a gene that is introduced into the host organism by gene transfer. 15 Foreign genes can comprise genes inserted into a non-native organism, genes introduced into a new location within the native host, or chimeric genes. The term "native nucleotide sequence" refers to a nucleotide sequence that is normally found in the host microorganism. 20 The term "non-native nucleotide sequence" refers to a nucleotide sequence that is not normally found in the host microorganism. The term "native polypeptide" refers to a polypeptide that is normally found in the host microorganism. The term "non-native polypeptide" refers to a polypeptide that is not 25 normally found in the host microorganism. The terms "encoding" and "coding" are used interchangeably herein and refer to the process by which a gene, through the mechanisms of transcription and translation, produces an amino acid sequence. The term "coding sequence" refers to a nucleotide sequence that 30 codes for a specific amino acid sequence. "Suitable regulatory sequences" refer to nucleotide sequences located upstream (5' non-coding sequences), within, or downstream 16 (3' non-coding sequences) of a coding sequence, and which influence the transcription, RNA processing or stability, or translation of the associated coding sequence. Regulatory sequences may include promoters, enhancers, silencers, 5' untranslated leader sequence (e.g., between the 5 transcription start site and the translation initiation codon), introns, polyadenylation recognition sequences, RNA processing sites, effector binding sites and stem-loop structures. The term "expression cassette" refers to a fragment of DNA comprising the coding sequence of a selected gene and regulatory io sequences preceding (5' non-coding sequences) and following (3' non coding sequences) the coding sequence that are required for expression of the selected gene product. Thus, an expression cassette is typically composed of: 1) a promoter sequence; 2) a coding sequence (i.e., ORF) and, 3) a 3' untranslated region (e.g., a terminator) that, in eukaryotes, 15 usually contains a polyadenylation site. The expression cassette(s) is usually included within a vector, to facilitate cloning and transformation. Different organisms, including bacteria, yeast, and fungi, can be transformed with different expression cassettes as long as the correct regulatory sequences are used for each host. 20 "Transformation" refers to the transfer of a nucleic acid molecule into a host organism, resulting in genetically stable inheritance. The nucleic acid molecule may be a plasmid that replicates autonomously, for example, or it may integrate into the genome of the host organism. Host organisms transformed with the nucleic acid fragments are referred to as 25 "recombinant" or "transformed" organisms or "transformants". "Stable transformation" refers to the transfer of a nucleic acid fragment into a genome of a host organism, including both nuclear and organellar genomes, resulting in genetically stable inheritance. In contrast, "transient transformation" refers to the transfer of a nucleic acid fragment into the 30 nucleus, or DNA-containing organelle, of a host organism resulting in gene expression without integration or stable inheritance. 17 "Codon degeneracy" refers to the nature in the genetic code permitting variation of the nucleotide sequence without effecting the amino acid sequence of an encoded polypeptide. The skilled artisan is well aware of the "codon-bias" exhibited by a specific host cell in usage of 5 nucleotide codons to specify a given amino acid. Therefore, when synthesizing a gene for improved expression in a host cell, it is desirable to design the gene such that its frequency of codon usage approaches the frequency of preferred codon usage of the host cell. The terms "subfragment that is functionally equivalent" and 10 "functionally equivalent subfragment" are used interchangeably herein. These terms refer to a portion or subsequence of an isolated nucleic acid fragment in which the ability to alter gene expression or produce a certain phenotype is retained whether or not the fragment or subfragment encodes an active enzyme. Chimeric genes can be designed for use in 15 suppression by linking a nucleic acid fragment or subfragment thereof, whether or not it encodes an active enzyme, in the sense or antisense orientation relative to a promoter sequence. The term "conserved domain" or "motif" means a set of amino acids conserved at specific positions along an aligned sequence of evolutionarily 20 related proteins. While amino acids at other positions can vary between homologous proteins, amino acids that are highly conserved at specific positions indicate amino acids that are essential in the structure, the stability, or the activity of a protein. The terms "substantially similar" and "corresponds substantially" are 25 used interchangeably herein. They refer to nucleic acid fragments wherein changes in one or more nucleotide bases do not affect the ability of the nucleic acid fragment to mediate gene expression or produce a certain phenotype. These terms also refer to modifications of the nucleic acid fragments of the instant invention such as deletion or insertion of one 30 or more nucleotides that do not substantially alter the functional properties of the resulting nucleic acid fragment relative to the initial, unmodified fragment. It is therefore understood, as those skilled in the art will 18 appreciate, that the invention encompasses more than the specific exemplary sequences. Moreover, the skilled artisan recognizes that substantially similar nucleic acid sequences encompassed by this invention are also defined by their ability to hybridize (under moderately 5 stringent conditions, e.g., 0.5X SSC (standard sodium citrate), 0.1% SDS (sodium dodecyl sulfate), 60 0C) with the sequences exemplified herein, or to any portion of the nucleotide sequences disclosed herein and which are functionally equivalent to any of the nucleic acid sequences disclosed herein. Stringency conditions can be adjusted to screen for moderately io similar fragments, such as homologous sequences from distantly related organisms, to highly similar fragments, such as genes that duplicate functional enzymes from closely related organisms. Post-hybridization washes determine stringency conditions. The term "selectively hybridizes" includes reference to 15 hybridization, under stringent hybridization conditions, of a nucleic acid sequence to a specified nucleic acid target sequence to a detectably greater degree (e.g., at least 2-fold over background) than its hybridization to non-target nucleic acid sequences and to the substantial exclusion of non-target nucleic acids. Selectively hybridizing sequences are two 20 nucleotide sequences wherein the complement of one of the nucleotide sequences typically has about at least 80% sequence identity, or 90% sequence identity, up to and including 100% sequence identity (i.e., fully complementary) to the other nucleotide sequence. The term "stringent conditions" or "stringent hybridization 25 conditions" includes reference to conditions under which a probe will selectively hybridize to its target sequence. Probes are typically single stranded nucleic acid sequences which are complementary to the nucleic acid sequences to be detected. Probes are "hybridizable" to the nucleic acid sequence to be detected. Generally, a probe is less than about 1000 30 nucleotides in length, optionally less than 500 nucleotides in length. Hybridization methods are well defined. Typically the probe and sample are mixed under conditions which will permit nucleic acid 19 hybridization. This involves contacting the probe and sample in the presence of an inorganic or organic salt under the proper concentration and temperature conditions. Optionally a chaotropic agent may be added. Nucleic acid hybridization is adaptable to a variety of assay formats. One 5 of the most suitable is the sandwich assay format. A primary component of a sandwich-type assay is a solid support. The solid support has adsorbed to it or covalently coupled to it an immobilized nucleic acid probe that is unlabeled and complementary to one portion of the sequence. Stringent conditions are sequence-dependent and will be different io in different circumstances. By controlling the stringency of the hybridization and/or washing conditions, target sequences can be identified which are 100% complementary to the probe (homologous probing). Alternatively, stringency conditions can be adjusted to allow some mismatching in sequences so that lower degrees of similarity are 15 detected (heterologous probing). Typically, stringent conditions will be those in which the salt concentration is less than about 1.5 M Na ion, typically about 0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30 0C for short probes (e.g., 10 to 50 nucleotides) and at 20 least about 60 0C for long probes (e.g., greater than 50 nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. Exemplary low stringency conditions include hybridization with a buffer solution of 30 to 35% formamide, 1 M NaCI, 1% SDS (sodium dodecyl sulfate) at 37 C, and a 25 wash in 1X to 2X SSC (20X SSC = 3.0 M NaCI/0.3 M trisodium citrate) at 50 to 55 0C. Exemplary moderate stringency conditions include hybridization in 40 to 45% formamide, 1 M NaCI, 1% SDS at 37 C, and a wash in 0.5X to 1X SSC at 55 to 60 C. Exemplary high stringency conditions include hybridization in 50% formamide, 1 M NaCI, 1% SDS at 30 37 C, and a wash in 0.1X SSC at 60 to 65 C. Specificity is typically the function of post-hybridization washes, the critical factors being the ionic strength and temperature of the final wash 20 solution. For DNA-DNA hybrids, the thermal melting point (Tm) can be approximated from the equation of Meinkoth et al., Anal. Biochem. 138:267-284 (1984): Tm = 81.5 0C + 16.6 (log M) + 0.41 (%GC) - 0.61 (% form) - 500/L; where M is the molarity of monovalent cations, %GC is the 5 percentage of guanosine and cytosine nucleotides in the DNA, % form is the percentage of formamide in the hybridization solution, and L is the length of the hybrid in base pairs. The Tm is the temperature (under defined ionic strength and pH) at which 50% of a complementary target sequence hybridizes to a perfectly matched probe. Tm is reduced by 10 about 1 0C for each 1% of mismatching; thus, Tm, hybridization and/or wash conditions can be adjusted to hybridize to sequences of the desired identity. For example, if sequences with >90% identity are sought, the Tm can be decreased 10 0C. Generally, stringent conditions are selected to be about 5 0C lower than Tm for the specific sequence and its complement 15 at a defined ionic strength and pH. However, severely stringent conditions can utilize a hybridization and/or wash at 1, 2, 3, or 4 0C lower than the Tm; moderately stringent conditions can utilize a hybridization and/or wash at 6, 7, 8, 9, or 10 0C lower than the Tm; low stringency conditions can utilize a hybridization and/or wash at 11, 12, 13, 14, 15, or 20 0C lower than the 20 Tm. Using the equation, hybridization and wash compositions, and desired Tm, those of ordinary skill will understand that variations in the stringency of hybridization and/or wash solutions are inherently described. If the desired degree of mismatching results in a Tm of less than 45 0C (aqueous solution) or 32 0C (formamide solution) it is preferred to increase the SSC 25 concentration so that a higher temperature can be used. An extensive guide to the hybridization of nucleic acids is found in Tijssen, Laboratory Techniques in Biochemistry and Molecular Biology--Hybridization with Nucleic Acid Probes, Part I, Chapter 2 "Overview of principles of hybridization and the strategy of nucleic acid probe assays", Elsevier, New 30 York (1993); and Current Protocols in Molecular Biology, Chapter 2, Ausubel et al., Eds., Greene Publishing and Wiley-Interscience, New York (1995). Hybridization and/or wash conditions can be applied for at least 21 10, 30, 60, 90, 120, or 240 minutes. "Sequence identity" or "identity" in the context of nucleic acid or polypeptide sequences refers to the nucleic acid bases or amino acid residues in two sequences that are the same when aligned for maximum 5 correspondence over a specified comparison window. Thus, "percentage of sequence identity" refers to the value determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may comprise additions io or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, 15 dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the results by 100 to yield the percentage of sequence identity. Useful examples of percent sequence identities include, but are not limited to, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, or any integer percentage from 50% to 100%. 20 These identities can be determined using any of the programs described herein. Sequence alignments and percent identity or similarity calculations may be determined using a variety of comparison methods designed to detect homologous sequences including, but not limited to, the MegAlign T M 25 program of the LASERGENE bioinformatics computing suite (DNASTAR Inc., Madison, WI). Within the context of this application it will be understood that where sequence analysis software is used for analysis, that the results of the analysis will be based on the "default values" of the program referenced, unless otherwise specified. As used herein "default 30 values" will mean any set of values or parameters that originally load with the software when first initialized. The "Clustal V method of alignment" corresponds to the alignment 22 method labeled Clustal V (described by Higgins and Sharp, CAB/OS. 5:151-153 (1989); Higgins, D.G. et al., Comput. App. Biosci. 8:189-191 (1992)) and found in the MegAlign T M program of the LASERGENE bioinformatics computing suite (DNASTAR Inc., Madison, WI). For 5 multiple alignments, the default values correspond to GAP PENALTY=1 0 and GAP LENGTH PENALTY=1 0. Default parameters for pairwise alignments and calculation of percent identity of protein sequences using the Clustal V method are KTUPLE=1, GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5. For nucleic acids these parameters are 10 KTUPLE=2, GAP PENALTY=5, WINDOW=4 and DIAGONALS SAVED=4. After alignment of the sequences using the Clustal V program, it is possible to obtain a "percent identity" by viewing the "sequence distances" table in the same program. The "Clustal W method of alignment" corresponds to the alignment 15 method labeled Clustal W (described by Higgins and Sharp, supra; Higgins, D.G. et al., supra) and found in the MegAlign T M v6.1 program of the LASERGENE bioinformatics computing suite (DNASTAR Inc., Madison, WI). Default parameters for multiple alignment correspond to GAP PENALTY=1 0, GAP LENGTH PENALTY=0.2, Delay Divergen 20 Seqs(%)=30, DNA Transition Weight=0.5, Protein Weight Matrix=Gonnet Series, DNA Weight Matrix=IUB. After alignment of the sequences using the Clustal W program, it is possible to obtain a "percent identity" by viewing the "sequence distances" table in the same program. "BLASTN method of alignment" is an algorithm provided by the 25 National Center for Biotechnology Information (NCBI) to compare nucleotide sequences using default parameters. It is well understood by one skilled in the art that many levels of sequence identity are useful in identifying polypeptides, from other species, wherein such polypeptides have the same or similar function or 30 activity. Useful examples of percent identities include, but are not limited to, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, or any integer percentage from 50% to 100%. Indeed, any integer amino acid 23 identity from 50% to 100% may be useful in describing the present invention, such as 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 5 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%. Also, of interest is any full-length or partial complement of this isolated nucleotide fragment. Thus, the invention encompasses more than the specific exemplary nucleotide sequences disclosed herein. For example, alterations in the io gene sequence which reflect the degeneracy of the genetic code are contemplated. Also, it is well known in the art that alterations in a gene which result in the production of a chemically equivalent amino acid at a given site, but do not affect the functional properties of the encoded protein are common. Substitutions are defined for the discussion herein 15 as exchanges within one of the following five groups: 1. Small aliphatic, nonpolar or slightly polar residues: Ala, Ser, Thr (Pro, Gly); 2. Polar, negatively charged residues and their amides: Asp, Asn, Glu, Gin; 20 3. Polar, positively charged residues: His, Arg, Lys; 4. Large aliphatic, nonpolar residues: Met, Leu, lie, Val (Cys); and 5. Large aromatic residues: Phe, Tyr, Trp. Thus, a codon for the amino acid alanine, a hydrophobic amino acid, may 25 be substituted by a codon encoding another less hydrophobic residue (such as glycine) or a more hydrophobic residue (such as valine, leucine, or isoleucine). Similarly, changes which result in substitution of one negatively charged residue for another (such as aspartic acid for glutamic acid) or one positively charged residue for another (such as lysine for 30 arginine) can also be expected to produce a functionally equivalent product. In many cases, nucleotide changes which result in alteration of the N-terminal and C-terminal portions of the protein molecule would also 24 not be expected to alter the activity of the protein. Each of the proposed modifications is well within the routine skill in the art, as is determination of retention of biological activity of the encoded products. Moreover, the skilled artisan recognizes that substantially 5 similar sequences encompassed by this invention are also defined by their ability to hybridize under stringent conditions, as defined above. Preferred substantially similar nucleic acid fragments of the instant invention are those nucleic acid fragments whose nucleotide sequences are at least 70% identical to the nucleotide sequence of the nucleic acid io fragments reported herein. More preferred nucleic acid fragments are at least 90% identical to the nucleotide sequence of the nucleic acid fragments reported herein. Most preferred are nucleic acid fragments that are at least 95% identical to the nucleotide sequence of the nucleic acid fragments reported herein. 15 A "substantial portion" of an amino acid or nucleotide sequence is that portion comprising enough of the amino acid sequence of a polypeptide or the nucleotide sequence of a gene to putatively identify that polypeptide or gene, either by manual evaluation of the sequence by one skilled in the art, or by computer-automated sequence comparison and 20 identification using algorithms such as BLAST (Basic Local Alignment Search Tool; Altschul, S. F., et al., J. Mol. Biol., 215:403-410 (1993)). In general, a sequence of ten or more contiguous amino acids or thirty or more nucleotides is necessary in order to putatively identify a polypeptide or nucleic acid sequence as homologous to a known protein or gene. 25 Moreover, with respect to nucleotide sequences, gene-specific oligonucleotide probes comprising 20-30 contiguous nucleotides may be used in sequence-dependent methods of gene identification (e.g., Southern hybridization) and isolation (e.g., in situ hybridization of bacterial colonies or bacteriophage plaques). In addition, short oligonucleotides of 30 12-15 bases may be used as amplification primers in PCR in order to obtain a particular nucleic acid fragment comprising the primers. Accordingly, a "substantial portion" of a nucleotide sequence comprises 25 enough of the sequence to specifically identify and/or isolate a nucleic acid fragment comprising the sequence. The instant specification teaches the complete amino acid and nucleotide sequence encoding particular proteins. The skilled artisan, having the benefit of the sequences as 5 reported herein, may now use all or a substantial portion of the disclosed sequences for purposes known to those skilled in this art. The term "complementary" describes the relationship between two sequences of nucleotide bases that are capable of Watson-Crick base pairing when aligned in an anti-parallel orientation. For example, with io respect to DNA, adenosine is capable of base-pairing with thymine and cytosine is capable of base-pairing with guanine. Accordingly, the instant invention may make use of isolated nucleic acid molecules that are complementary to the complete sequences as reported in the accompanying Sequence Listing and the specification as well as those 15 substantially similar nucleic acid sequences. The term "isolated" refers to a polypeptide or nucleotide sequence that is removed from at least one component with which it is naturally associated. "Promoter" refers to a DNA sequence capable of controlling the 20 expression of a coding sequence or functional RNA. The promoter sequence consists of proximal and more distal upstream elements, the latter elements often referred to as enhancers. Accordingly, an "enhancer" is a DNA sequence that can stimulate promoter activity, and may be an innate element of the promoter or a heterologous element inserted to 25 enhance the level or tissue-specificity of a promoter. Promoters may be derived in their entirety from a native gene, or be composed of different elements derived from different promoters found in nature, or even comprise synthetic DNA segments. It is understood by those skilled in the art that different promoters may direct the expression of a gene in different 30 tissues or cell types, or at different stages of development, or in response to different environmental conditions. It is further recognized that since in most cases the exact boundaries of regulatory sequences have not been 26 completely defined, DNA fragments of some variation may have identical promoter activity. Promoters that cause a gene to be expressed in most cell types at most times are commonly referred to as "constitutive promoters". 5 "3' non-coding sequences", "transcription terminator" and "termination sequences" are used interchangeably herein and refer to DNA sequences located downstream of a coding sequence, including polyadenylation recognition sequences and other sequences encoding regulatory signals capable of affecting mRNA processing or gene 10 expression. The polyadenylation signal is usually characterized by affecting the addition of polyadenylic acid tracts to the 3' end of the mRNA precursor. The term "operably linked" refers to the association of nucleic acid sequences on a single nucleic acid fragment so that the function of one is 15 affected by the other. For example, a promoter is operably linked with a coding sequence when it is capable of affecting the expression of that coding sequence (i.e., the coding sequence is under the transcriptional control of the promoter). Coding sequences can be operably linked to regulatory sequences in a sense or antisense orientation. In another 20 example, the complementary RNA regions of the invention can be operably linked, either directly or indirectly, 5' to the target mRNA, or 3' to the target mRNA, or within the target mRNA, or a first complementary region is 5' and its complement is 3' to the target mRNA. Standard recombinant DNA and molecular cloning techniques used 25 herein are well known in the art and are described more fully in Sambrook, J., Fritsch, E.F. and Maniatis, T. Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory: Cold Spring Harbor, NY (1989). Transformation methods are well known to those skilled in the art and are described infra. 30 "PCR" or "polymerase chain reaction" is a technique for the synthesis of large quantities of specific DNA segments and consists of a series of repetitive cycles (Perkin Elmer Cetus Instruments, Norwalk, CT). 27 Typically, the double-stranded DNA is heat denatured, the two primers complementary to the 3' boundaries of the target segment are annealed at low temperature and then extended at an intermediate temperature. One set of these three consecutive steps is referred to as a "cycle". 5 A "plasmid" or "vector" is an extra chromosomal element often carrying genes that are not part of the central metabolism of the cell, and usually in the form of circular double-stranded DNA fragments. Such elements may be autonomously replicating sequences, genome integrating sequences, phage or nucleotide sequences, linear or circular, io of a single- or double-stranded DNA or RNA, derived from any source, in which a number of nucleotide sequences have been joined or recombined into a unique construction which is capable of introducing an expression cassette(s) into a cell. The term "genetically altered" refers to the process of changing 15 hereditary material by genetic engineering, transformation and/or mutation. The term "recombinant" refers to an artificial combination of two otherwise separated segments of sequence, e.g., by chemical synthesis or by the manipulation of isolated segments of nucleic acids by genetic 20 engineering techniques. "Recombinant" also includes reference to a cell or vector, that has been modified by the introduction of a heterologous nucleic acid or a cell derived from a cell so modified, but does not encompass the alteration of the cell or vector by naturally occurring events (e.g., spontaneous mutation, natural transformation, natural transduction, 25 natural transposition) such as those occurring without deliberate human intervention. The terms "recombinant construct", "expression construct", "chimeric construct", "construct", and "recombinant DNA construct", are used interchangeably herein. A recombinant construct comprises an 30 artificial combination of nucleic acid fragments, e.g., regulatory and coding sequences that are not found together in nature. For example, a recombinant construct may comprise regulatory sequences and coding 28 sequences that are derived from different sources, or regulatory sequences and coding sequences derived from the same source, but arranged in a manner different than that found in nature. Such a construct may be used by itself or may be used in conjunction with a vector. If a 5 vector is used, then the choice of vector is dependent upon the method that will be used to transform host cells as is well known to those skilled in the art. For example, a plasmid vector can be used. The skilled artisan is well aware of the genetic elements that must be present on the vector in order to successfully transform, select and propagate host cells io comprising any of the isolated nucleic acid fragments of the invention. The skilled artisan will also recognize that different independent transformation events may result in different levels and patterns of expression (Jones et al., EMBO J. 4:2411-2418 (1985); De Almeida et al., Mol. Gen. Genetics 218:78-86 (1989)), and thus that multiple events may 15 need be screened in order to obtain lines displaying the desired expression level and pattern. Such screening may be accomplished by Southern analysis of DNA, Northern analysis of mRNA expression, immunoblotting analysis of protein expression, or phenotypic analysis, among others. 20 The term "expression", as used herein, refers to the production of a functional end-product (e.g., an mRNA or a protein [either precursor or mature]). The term "introduced" means providing a nucleic acid (e.g., expression construct) or protein into a cell. Introduced includes reference 25 to the incorporation of a nucleic acid into a eukaryotic or prokaryotic cell where the nucleic acid may be incorporated into the genome of the cell, and includes reference to the transient provision of a nucleic acid or protein to the cell. Introduced includes reference to stable or transient transformation methods, as well as sexually crossing. Thus, "introduced" 30 in the context of inserting a nucleic acid fragment (e.g., a recombinant construct/expression construct) into a cell, means "transfection" or "transformation" or "transduction" and includes reference to the 29 incorporation of a nucleic acid fragment into a eukaryotic or prokaryotic cell where the nucleic acid fragment may be incorporated into the genome of the cell (e.g., chromosome, plasmid, plastid or mitochondrial DNA), converted into an autonomous replicon, or transiently expressed (e.g., 5 transfected mRNA). The term "homologous" refers to proteins or polypeptides of common evolutionary origin with similar catalytic function. The invention may include bacteria producing homologous proteins via recombinant technology. 10 Disclosed herein are recombinant bacteria comprising in their genome or on at least one recombinant construct: one or more nucleotide sequences encoding a polypeptide or a polypeptide complex having sucrose transporter activity; a nucleotide sequence encoding a polypeptide having fructokinase activity; and a nucleotide sequence 15 encoding a polypeptide having sucrose hydrolase activity. These nucleotide sequences are each operably linked to the same or a different promoter. These recombinant bacteria are capable of metabolizing sucrose to produce glycerol and/or glycerol-derived products such as 1,3 propanediol and 3-hydroxypropionic acid. Bacterial strains capable of 20 producing glycerol and/or glycerol-derived products are highly engineered strains, as described herein below. Suitable host bacteria for use in the construction of the recombinant bacteria disclosed herein include, but are not limited to organisms of the genera: Escherichia, Streptococcus, Agrobacterium, Bacillus, 25 Corynebacterium, Lactobacillus, Clostridium, Gluconobacter, Citrobacter, Enterobacter, Klebsiella, Aerobacter, Methylobacter, Salmonella, Streptomyces, and Pseudomonas. In one embodiment the host bacterium is selected from the genera: Escherichia, Klebsiella, Citrobacter, and Aerobacter. 30 In another embodiment, the host bacterium is Escherichia coli. In some embodiments, the host bacterium is PTS minus. In these embodiments, the host bacterium is PTS minus in its native state, or may 30 be rendered PTS minus through inactivation of a PTS gene as described below. In production microorganisms, it is sometimes desirable to unlink the transport of sugars and the use of phosphoenolpyruvate (PEP) for 5 phosphorylation of the sugars being transported. The term "down-regulated" refers to reduction in, or abolishment of, the activity of active protein(s), as compared to the activity of the wildtype protein(s). The PTS may be inactivated (resulting in a "PTS minus" organism) by down-regulating expression of one or more of the 10 endogenous genes encoding the proteins required in this type of transport. Down-regulation typically occurs when one or more of these genes has a "disruption", referring to an insertion, deletion, or targeted mutation within a portion of that gene, that results in either a complete gene knockout such that the gene is deleted from the genome and no protein is translated 15 or a protein has been translated such that it has an insertion, deletion, amino acid substitution or other targeted mutation. The location of the disruption in the protein may be, for example, within the N-terminal portion of the protein or within the C-terminal portion of the protein. The disrupted protein will have impaired activity with respect to the protein that was not 20 disrupted, and can be non-functional. Down-regulation that results in low or lack of expression of the protein, could also result via manipulating the regulatory sequences, transcription and translation factors and/or signal transduction pathways or by use of sense, antisense or RNAi technology, etc. 25 Sucrose transporter polypeptides or polypeptide complexes are polypeptides or polypeptide complexes that are capable of mediating the transport of sucrose into microbial cells. Sucrose transport polypeptides and polypeptide complexes are known, as described above. Examples of polypeptides having sucrose transporter activity include, but are not limited 30 to, CscB from E. co/iwild-type strain EC3132 (set forth in SEQ ID NO:24), encoded by gene cscB (coding sequence set forth in SEQ ID NO:23); CscB from E. coli ATC 3281 (set forth in SEQ ID NO:26), encoded by 31 gene cscB (coding sequence set forth in SEQ ID NO:25); and CscB from Bifidobacterium lactis (set forth in SEQ ID NO:28), encoded by gene cscB (coding sequence set forth in SEQ ID NO:27). Examples of polypeptide complexes having sucrose transporter activity include, but are not limited 5 to, the sucrose ABC-type transporter complex from Streptococcus pneumoniae strain TIGR4 comprising three polypeptide subunits set forth in SEQ ID NOs:30, 32, and 34, encoded by genes susT1 (coding sequence set forth in SEQ ID NO:29), susT2 (coding sequence set forth in SEQ ID NO:31), and susX (coding sequence set forth in SEQ ID NO: 33); io and the maltose transporter complex of Streptococcus mutans comprising four polypeptide subunits set forth in SEQ ID NOs:36, 38, 40, and 42, encoded by genes malE (coding sequence set forth in SEQ ID NO:35), ma/F (coding sequence set forth in SEQ ID NO:37), maIG (coding sequence set forth in SEQ ID NO:39), and malK (coding sequence set 15 forth in SEQ ID NO:41), respectively. In one embodiment, the polypeptide having sucrose transporter activity has at least 95% sequence identity, based on the Clustal V method of alignment, to an amino acid sequence as set forth in SEQ ID NO:24, SEQ ID NO:26, or SEQ ID NO:28. 20 In another embodiment, the polypeptide complex having sucrose transporter activity comprises: a first subunit having at least 95% sequence identity, based on a Clustal V method of alignment, when compared to an amino acid sequence as set forth in SEQ ID NO:30; a second subunit having at least 95% sequence identity, based on a Clustal 25 V method of alignment, when compared to an amino acid sequence as set forth in SEQ ID NO:32; and a third subunit having at least 95% sequence identity, based on a Clustal V method of alignment, when compared to an amino acid sequence as set forth in SEQ ID NO:34. In another embodiment, the polypeptide complex having sucrose 30 transporter activity comprises: a first subunit having at least 95% sequence identity, based on a Clustal V method of alignment, when compared to an amino acid sequence as set forth in SEQ ID NO:36; a 32 second subunit having at least 95% sequence identity, based on a Clustal V method of alignment, when compared to an amino acid sequence as set forth in SEQ ID NO:38; a third subunit having at least 95% sequence identity, based on a Clustal V method of alignment, when compared to an 5 amino acid sequence as set forth in SEQ ID NO:40; and a fourth subunit having at least 95% sequence identity, based on a Clustal V method of alignment, when compared to an amino acid sequence as set forth in SEQ ID NO:42. In another embodiment, the polypeptide having sucrose transporter io activity corresponds substantially to the amino acid sequence set forth in SEQ ID NO:26. Polypeptides having fructokinase activity include fructokinases (designated EC 2.7.1.4) and various hexose kinases having fructose phosphorylating activity (EC 2.7.1.3 and EC 2.7.1.1). Fructose 15 phosphorylating activity may be exhibited by hexokinases and ketohexokinases. Representative genes encoding polypeptides from a variety of microorganisms, which may be used to construct the recombinant bacteria disclosed herein, are listed in Table 1. One skilled in the art will know that proteins that are substantially similar to a protein 20 which is able to phosphorylate fructose (such as encoded by the genes listed in Table 1) may also be used. 33 Table 1 Sequences Encoding Enzymes with Fructokinase Activity Nucleotide Protein SEQ ID SEQ ID Source Gene Name EC Number NO: NO: Agrobacterium 43 44 tumefaciens scrK (fructoki nase) 2.7.1.4 Streptococcus 45 46 mutans scrK (fructokinase) 2.7.1.4 Escherichia coli scrK (fructokinase 2.7.1.4 84 85 Klebsiella 86 87 pneumoniae scrK (fructokinase 2.7.1.4 Escherichia coli cscK (fructokinase) 2.7.1.4 47 48 Enterococcus faecalis cscK (fructokinase) 2.7.1.4 49 50 Saccharomyces 51 52 cerevisiae HXK1 (hexokinase) 2.7.1.1 Saccharomyces 53 54 cerevisiae HXK2 (hexokinase) 2.7.1.1 In one embodiment, the polypeptide having fructokinase activity has 5 at least 95% sequence identity, based on the Clustal V method of alignment, to an amino acid sequence as set forth in SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:85, or SEQ ID NO:87. In another embodiment, the polypeptide having fructokinase activity 10 corresponds substantially to the sequence set forth in SEQ ID NO:48 Polypeptides having sucrose hydrolase activity have the ability to catalyze the hydrolysis of sucrose to produce fructose and glucose. Polypeptides having sucrose hydrolase activity are known, as described above, and include, but are not limited to CscA from E. co/iwild-type strain 15 EC3132 (set forth in SEQ ID NO:56), encoded by gene cscA (coding sequence set forth in SEQ ID NO:55), CscA from E. coli ATC1 3821 (set forth in SEQ ID NO:58), encoded by gene cscA (coding sequence set forth in SEQ ID NO:57); BfrA from Bifidobacterium lactis strain DSM 10 14 0 T (set forth in SEQ ID NO:60), encoded by gene bfrA (coding sequence set 20 forth in SEQ ID NO:59); Suc2p from Saccharomyces cerevisiae (set forth 34 in SEQ ID NO:62), encoded by gene SUC2 (coding sequence set forth in SEQ ID NO:61); ScrB from Corynebacterium glutamicum (set forth in SEQ ID NO:64), encoded by gene scrB (coding sequence set forth in SEQ ID NO:63); sucrose phosphorylase from Leuconostoc mesenteroides DSM 5 20193 (set forth in SEQ ID NO:66), coding sequence of encoding gene set forth in SEQ ID NO:65; and sucrose phosphorylase from Bifidobacterium adolescents DSM 20083 (set forth in SEQ ID NO:68), encoded by gene sucP (coding sequence set forth in SEQ ID NO:67). In one embodiment, the polypeptide having sucrose hydrolase 10 activity has at least 95% sequence identity, based on the Clustal V method of alignment, to an amino acid sequence as set forth in SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, or SEQ ID NO:68. In another embodiment, the polypeptide having sucrose hydrolase 15 activity corresponds substantially to the amino acid sequence set forth in SEQ ID NO:58. The coding sequence of genes encoding polypeptides or polypeptide complexes having sucrose transporter activity, polypeptides having fructokinase activity, and polypeptides having sucrose hydrolase 20 activity may be used to isolate nucleotide sequences encoding homologous polypeptides from the same or other microbial species. For example, homologs of the genes may be identified using sequence analysis software, such as BLASTN, to search publically available nucleic acid sequence databases. Additionally, the isolation of homologous genes 25 using sequence-dependent protocols is well known in the art. Examples of sequence-dependent protocols include, but are not limited to, methods of nucleic acid hybridization, and methods of DNA and RNA amplification as exemplified by various uses of nucleic acid amplification technologies (e.g. polymerase chain reaction (PCR), Mullis et al., U.S. Patent No. 30 4,683,202; ligase chain reaction (LCR), Tabor, S. et al., Proc. Acad. Sci. USA 82, 1074, 1985); or strand displacement amplification (SDA), Walker, et al., Proc. Nat/. Acad. Sci. U.S.A., 89: 392, (1992)). For example, the 35 nucleotide sequence encoding the polypeptides described above may be employed as a hybridization probe for the identification of homologs. One of ordinary skill in the art will appreciate that genes encoding these polypeptides isolated from other sources may also be used in the 5 recombinant bacteria disclosed herein. Additionally, variations in the nucleotide sequences encoding the polypeptides may be made without affecting the amino acid sequence of the encoded polypeptide due to codon degeneracy, and that amino acid substitutions, deletions or additions that produce a substantially similar protein may be included in io the encoded protein. The nucleotide sequences encoding polypeptides or polypeptide complexes having sucrose transporter activity, polypeptides having fructokinase activity, and polypeptides having sucrose hydrolase activity may be isolated using PCR (see, e.g., U.S. Patent No. 4,683,202) and 15 primers designed to bound the desired sequence, if this sequence is known. Other methods of gene isolation are well known to one skilled in the art such as by using degenerate primers or heterologous probe hybridization. The nucleotide sequences can also be chemically synthesized or purchased from vendors such as DNA2.0 Inc. (Menlo 20 Park, CA). Additionally, the entire csc operon may be isolated from the genomic DNA of E. coli strain ATCC1 3281, as described in detail in Example 1 herein. Expression of the polypeptides may be effected using one of many methods known to one skilled in the art. For example, the 25 nucleotide sequences encoding the polypeptides described above may be introduced into the bacterium on at least one multicopy plasmid, or by integrating one or more copies of the coding sequences into the host genome. The nucleotide sequences encoding the polypeptides may be introduced into the host bacterium separately (e.g., on separate 30 plasmids) or in any combination (e.g., on a single plasmid, as described in the Examples herein). If the host bacterium contains a gene encoding one of the polynucleotides, then only the remaining nucleotide 36 sequences need to be introduced into the bacterium. For example, if the host bacterium contains a nucleotide sequence encoding a polypeptide having fructokinase activity, only a nucleotide sequence encoding a polypeptide having sucrose transporter activity and a nucleotide 5 sequence encoding a polypeptide having sucrose hydrolase activity need to be introduced into the bacterium to enable sucrose utilization. The introduced coding regions that are either on a plasmid(s) or in the genome may be expressed from at least one highly active promoter. An integrated coding region may either be introduced as a part of a chimeric io gene having its own promoter, or it may be integrated adjacent to a highly active promoter that is endogenous to the genome or in a highly expressed operon. Suitable promoters include, but are not limited to, CYC1, HIS3, GAL1, GAL10, ADH1, PGK, PHO5, GAPDH, ADC1, TRP1, URA3, LEU2, ENO, and lac, ara, tet, trp, /PL, 1PR, T7, tac, and trc 15 (useful for expression in Escherichia co/i) as well as the amy, apr, npr promoters and various phage promoters useful for expression in Bacillus. The promoter may also be the Streptomyces lividans glucose isomerase promoter or a variant thereof, described by Payne et al. (U.S. Patent No. 7,132,527). 20 In one embodiment, the recombinant bacteria disclosed herein are capable of producing glycerol. Biological processes for the preparation of glycerol using carbohydrates or sugars are known in yeasts and in some bacteria, other fungi, and algae. Both bacteria and yeasts produce glycerol by converting glucose or other carbohydrates 25 through the fructose-1,6-bisphosphate pathway in glycolysis. In the method of producing glycerol disclosed herein, host bacteria may be used that naturally produce glycerol. In addition, bacteria may be engineered for production of glycerol and glycerol derivatives. The capacity for glycerol production from a variety of substrates may be 30 provided through the expression of the enzyme activities glycerol-3 phosphate dehydrogenase (G3PDH) and/or glycerol-3-phosphatase as described in U.S. Patent No. 7,005,291. Genes encoding these proteins 37 that may be used for expressing the enzyme activities in a host bacterium are described in U.S. Patent No. 7,005,291. Suitable examples of genes encoding polypeptides having glycerol-3-phosphate dehydrogenase activity include, but are not limited to, GPD1 from 5 Saccharomyces cerevisiae (coding sequence set forth in SEQ ID NO:1, encoded protein sequence set forth in SEQ ID NO:2) and GPD2from Saccharomyces cerevisiae (coding sequence set forth in SEQ ID NO:3, encoded protein sequence set forth in SEQ ID NO:4). Suitable examples of genes encoding polypeptides having glycerol-3 10 phosphatase activity include, but are not limited to, GPP1 from Saccharomyces cerevisiae (coding sequence set forth in SEQ ID NO:5, encoded protein sequence set forth in SEQ ID NO:6) and GPP2from Saccharomyces cerevisiae (coding sequence set forth in SEQ ID NO:7, encoded protein sequence set forth in SEQ ID NO:8). 15 Increased production of glycerol may be attained through reducing expression of target endogenous genes. Down-regulation of endogenous genes encoding glycerol kinase and glycerol dehydrogenase activities further enhance glycerol production as described in U.S. Patent No. 7,005,291. Increased channeling of carbon to glycerol may be 20 accomplished by reducing the expression of the endogenous gene encoding glyceraldehyde 3-phosphate dehydrogenase, as described in U.S. Patent No. 7,371,558. Down-regulation may be accomplished by using any method known in the art, for example, the methods described above for down-regulation of genes of the PTS system. 25 Glycerol provides a substrate for microbial production of useful products. Examples of such products, i.e., glycerol derivatives include, but are not limited to, 3-hydroxypropionic acid, methylglyoxal, 1,2-propanediol, and 1,3-propanediol. In another embodiment, the recombinant bacteria disclosed herein 30 are capable of producing 1,3-propanediol. The glycerol derivative 1,3-propanediol is a monomer having potential utility in the production of polyester fibers and the manufacture of polyurethanes and cyclic 38 compounds. 1,3-Propanediol can be produced by a single microorganism by bioconversion of a carbon substrate other than glycerol or dihydroxyacetone, as described in U.S. Patent No. 5,686,276. In this bioconversion, glycerol is produced from the carbon substrate, as 5 described above. Glycerol is converted to the intermediate 3 hydroxypropionaldehyde by a dehydratase enzyme, which can be encoded by the host bacterium or can be introduced into the host by recombination. The dehydratase can be glycerol dehydratase (E.C. 4.2.1.30), diol dehydratase (E.C. 4.2.1.28) or any other enzyme able to io catalyze this conversion. A suitable example of genes encoding the " " (alpha), " " (beta), and " " (gamma) subunits of a glycerol dehydratase include, but are not limited to dhaBl (coding sequence set forth in SEQ ID NO:9), dhaB2 (coding sequence set forth in SEQ ID NO:1 1), and dhaB3 (coding sequence set forth in SEQ ID NO:13), respectively, from Klebsiella 15 pneumoniae. The further conversion of 3-hydroxypropionaldehyde to 1,3 propandeiol can be catalyzed by 1,3-propanediol dehydrogenase (E.C. 1.1.1.202) or other alcohol dehydrogenases. A suitable example of a gene encoding a 1,3-propanediol dehydrogenase is dhaTfrom Klebsiella pneumoniae (coding sequence set forth in SEQ ID NO:69, encoded 20 protein sequence set forth in SEQ ID NO:70). Bacteria can be recombinantly engineered to provide more efficient production of glycerol and the glycerol derivative 1,3-propanediol. For example, U.S. Patent No. 7,005,291 discloses transformed microorganisms and a method for production of glycerol and 1,3 25 propanediol with advantages derived from expressing exogenous activities of one or both of glycerol-3-phosphate dehydrogenase and glycerol-3 phosphate phosphatase while disrupting one or both of endogenous activities glycerol kinase and glycerol dehydrogenase. U.S. Patent No. 6,013,494 describes a process for the production of 30 1,3-propanediol using a single microorganism comprising exogenous glycerol-3-phosphate dehydrogenase, glycerol-3-phosphate phosphatase, dehydratase, and 1,3-propanediol oxidoreductase (e.g., dhaT). U.S. 39 Patent No. 6,136,576 discloses a method for the production of 1,3 propanediol comprising a recombinant microorganism further comprising a dehydratase and protein X (later identified as being a dehydratase reactivation factor peptide). 5 U.S. Patent No. 6,514,733 describes an improvement to the process where a significant increase in titer (grams product per liter) is obtained by virtue of a non-specific catalytic activity (distinguished from 1,3-propanediol oxidoreductase encoded by dhaT) to convert 3 hydroxypropionaldehyde to 1,3-propanediol. Additionally, U.S. Patent No. 10 7,132,527 discloses vectors and plasmids useful for the production of 1,3 propanediol. Increased production of 1,3-propanediol may be achieved by further modifications to a host bacterium, including down-regulating expression of some target genes and up-regulating, expression of other target genes, as 15 described in U.S. Patent No. 7,371,558. For utilization of glucose as a carbon source in a PTS minus host, expression of glucokinase activity may be increased. Additional genes whose increased or up-regulated expression increases 1,3-propanediol production include genes encoding: 20 e phosphoenolpyruvate carboxylase typically characterized as EC 4.1.1.31 * cob(I)alamin adenosyltransferase, typically characterized as EC 2.5.1.17 * non-specific catalytic activity that is sufficient to catalyze the 25 interconversion of 3-HPA and 1,3-propanediol, and specifically excludes 1,3-propanediol oxidoreductase(s), typically these enzymes are alcohol dehydrogenases Genes whose reduced or down-regulated expression increases 1,3 propanediol production include genes encoding: 30 e aerobic respiration control protein * methylglyoxal synthase e acetate kinase 40 * phosphotransacetylase * aldehyde dehydrogenase A * aldehyde dehydrogenase B * triosephosphate isomerase 5 e phosphogluconate dehydratase In another embodiment, the recombinant bacteria disclosed herein are capable of producing 3-hydroxypropionic acid. 3-Hydroxypropionic acid has utility for specialty synthesis and can be converted to commercially important intermediates by known art in the chemical 10 industry, e.g., acrylic acid by dehydration, malonic acid by oxidation, esters by esterification reactions with alcohols, and 1,3-propanediol by reduction. 3-Hydroxypropionic acid may be produced biologically from a fermentable carbon source by a single microorganism, as described in copending and commonly owned U.S. Patent Application No. 61/187476. 15 In one representative biosynthetic pathway, a carbon substrate is converted to 3-hydroxypropionaldehyde, as described above for the production of 1,3-propanediol. The 3-hydroxypropionaldehyde is converted to 3-hydroxypropionic acid by an aldehyde dehydrogenase. Suitable examples of aldehyde dehydrogenases include, but are not 20 limited to, AIdB (SEQ ID NO:16), encoded by the E. coligene aldB (coding sequence set forth in SEQ ID NO:1 5); AldA (SEQ ID NO:1 8), encoded by the E. coigene aldA (coding sequence set forth in SEQ ID NO:17); and AIdH (SEQ ID NO:20), encoded by the E. coligene aldH (coding sequence asset forth in SEQ ID NO:19). 25 Many of the modifications described above to improve 1,3 propanediol production by a recombinant bacterium can also be made to improve 3-hydroxypropionic acid production. For example, the elimination of glycerol kinase prevents glycerol, formed from G3P by the action of G3P phosphatase, from being re-converted to G3P at the expense of ATP. 30 Also, the elimination of glycerol dehydrogenase (for example, g/dA) prevents glycerol, formed from DHAP by the action of NAD-dependent glycerol-3-phosphate dehydrogenase, from being converted to 41 dihydroxyacetone. Mutations can be directed toward a structural gene so as to impair or improve the activity of an enzymatic activity or can be directed toward a regulatory gene, including promoter regions and ribosome binding sites, so as to modulate the expression level of an 5 enzymatic activity. Up-regulation or down-regulation may be achieved by a variety of methods which are known to those skilled in the art. It is well understood that up-regulation or down-regulation of a gene refers to an alteration in the level of activity present in a cell that is derived from the protein io encoded by that gene relative to a control level of activity, for example, by the activity of the protein encoded by the corresponding (or non-altered) wild-type gene. Specific genes involved in an enzyme pathway may be up regulated to increase the activity of their encoded function(s). For 15 example, additional copies of selected genes may be introduced into the host cell on multicopy plasmids such as pBR322. Such genes may also be integrated into the chromosome with appropriate regulatory sequences that result in increased activity of their encoded functions. The target genes may be modified so as to be under the control of non 20 native promoters or altered native promoters. Endogenous promoters can be altered in vivo by mutation, deletion, and/or substitution. Alternatively, it may be useful to reduce or eliminate the expression of certain genes relative to a given activity level. Methods of down regulating (disrupting) genes are known to those of skill in the art. 25 Down-regulation can occur by deletion, insertion, or alteration of coding regions and/or regulatory (promoter) regions. Specific down regulations may be obtained by random mutation followed by screening or selection, or, where the gene sequence is known, by direct intervention by molecular biology methods known to those skilled in the art. A particularly 30 useful, but not exclusive, method to effect down-regulation is to alter promoter strength. Furthermore, down-regulation of gene expression may be used to 42 either prevent expression of the protein of interest or result in the expression of a protein that is non-functional. This may be accomplished for example, by 1) deleting coding regions and/or regulatory (promoter) regions, 2) inserting exogenous nucleic acid sequences into coding regions 5 and/regulatory (promoter) regions, and 3) altering coding regions and/or regulatory (promoter) regions (for example, by making DNA base pair changes). Specific disruptions may be obtained by random mutation followed by screening or selection, or, in cases where the gene sequences in known, specific disruptions may be obtained by direct intervention using 10 molecular biology methods know to those skilled in the art. A particularly useful method is the deletion of significant amounts of coding regions and/or regulatory (promoter) regions. Methods of altering recombinant protein expression are known to those skilled in the art, and are discussed in part in Baneyx, Curr. Opin. 15 Biotechnol. (1999) 10:411; Ross, et al., J. Bacteriol. (1998) 180:5375; deHaseth, et al., J. Bacteriol. (1998) 180:3019; Smolke and Keasling, Biotechnol. Bioeng. (2002) 80:762; Swartz, Curr. Opin. Biotech. (2001) 12:195; and Ma, et al., J. Bacteriol. (2002) 184:5733. Recombinant bacteria containing the necessary changes in gene 20 expression for metabolizing sucrose in the production of microbial products including glycerol and glycerol derivatives, as described above, may be constructed using techniques well known in the art, some of which are exemplified in the Examples herein. The construction of the recombinant bacteria disclosed herein may 25 be accomplished using a variety of vectors and transformation and expression cassettes suitable for the cloning, transformation and expression of coding regions that confer the ability to utilize sucrose in the production of glycerol and its derivatives in a suitable host microorganism. Suitable vectors are those which are compatible with the bacterium 30 employed. Suitable vectors can be derived, for example, from a bacterium, a virus (such as bacteriophage T7 or a M-1 3 derived phage), a cosmid, a yeast or a plant. Protocols for obtaining and using such vectors 43 are known to those skilled in the art (Sambrook et al., supra). Initiation control regions, or promoters, which are useful to drive expression of coding regions for the instant invention in the desired host bacterium are numerous and familiar to those skilled in the art. Virtually 5 any promoter capable of driving expression is suitable for use herein. For example, any of the promoters listed above may be used. Termination control regions may also be derived from various genes native to the preferred hosts. Optionally, a termination site may be unnecessary; however, it is most preferred if included. 10 For effective expression of the instant polypeptides, nucleotide sequences encoding the polypeptides are linked operably through initiation codons to selected expression control regions such that expression results in the formation of the appropriate messenger RNA. Particularly useful in the present invention are the vectors 15 pSYCO1 01, pSYCO1 03, pSYCO1 06, and pSYCO1 09, described in U.S. Patent No. 7,371,558, and pSYCO400/AGRO, described in U.S. Patent No. 7,524,660. The essential elements of these vectors are derived from the dha regulon isolated from Klebsiella pneumoniae and from Saccharomyces cerevisiae. Each vector contains the open reading 20 frames dhaBl, dhaB2, dhaB3, dhaX (coding sequence set forth in SEQ ID NO:71), orfX, DAR1, and GPP2 arranged in three separate operons. The nucleotide sequences of pSYCO1 01, pSYCO1 03, pSYCO1 06, pSYCO1 09, and pSYCO400/AGRO are set forth in SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, and SEQ ID NO:76, 25 respectively. The differences between the vectors are illustrated in the chart below [the prefix "p-" indicates a promoter; the open reading frames contained within each "( )" represent the composition of an operon]: pSYCO101 (SEQ ID NO:72): p-trc (Dar1_GPP2) in opposite orientation compared to the other 2 30 pathway operons, p-1.6 long GI (dhaBldhaB2_dhaB3_dhaX), and p-1.6 long GI (orfYorfXorfW). 44 pSYCO103 (SEQ ID NO:73): p-trc (Dar1_GPP2) same orientation compared to the other 2 pathway operons, p-1.5 long GI (dhaBldhaB2_dhaB3_dhaX), and 5 p-1.5 long GI (orfYorfXorfW). pSYCO106 (SEQ ID NO:74): p-trc (Dar1_GPP2) same orientation compared to the other 2 pathway operons, p-1.6 long GI (dhaBldhaB2_dhaB3_dhaX), and 10 p-1.6 long GI (orfYorfXorfW). pSYCO109 (SEQ ID NO:75): p-trc (Dar1_GPP2) same orientation compared to the other 2 pathway operons, p-1.6 long GI (dhaBldhaB2_dhaB3_dhaX), and 15 p-1.6 long GI (orfY orfX). pSYCO400/AGRO (SEQ ID NO:76): p-trc (Dar1_GPP2) same orientation compared to the other 2 pathway operons, p-1.6 long GI (dhaBldhaB2_dhaB3_dhaX), and 20 p-1.6 long GI (orfY orfX). p-1.20 short/long GI (scrK) opposite orientation compared to the pathway operons. Once suitable expression cassettes are constructed, they are used to transform appropriate host bacteria. Introduction of the cassette 25 containing the coding regions into the host bacterium may be accomplished by known procedures such as by transformation (e.g., using calcium-permeabilized cells, or electroporation) or by transfection using a recombinant phage virus (Sambrook et al., supra). Expression cassettes may be maintained on a stable plasmid in a host cell. In addition, 30 expression cassettes may be integrated into the genome of the host bacterium through homologous or random recombination using vectors and methods well known to those skilled in the art. Site-specific 45 recombination systems may also be used for genomic integration of expression cassettes. In addition to the cells exemplified, cells having single or multiple mutations specifically designed to enhance the production of microbial 5 products including glycerol and/or its derivatives may also be used. Cells that normally divert a carbon feed stock into non-productive pathways, or that exhibit significant catabolite repression may be mutated to avoid these phenotypic deficiencies. Methods of creating mutants are common and well known in the art. 10 A summary of some methods is presented in U.S. Patent No. 7,371,558. Specific methods for creating mutants using radiation or chemical agents are well documented in the art. See, for example, Thomas D. Brock in Biotechnology: A Textbook of Industrial Microbiology, Second Edition (1989) Sinauer Associates, Inc., Sunderland, MA., or Deshpande, Mukund 15 V., Appl. Biochem. Biotechnol. 36, 227 (1992). After mutagenesis has occurred, mutants having the desired phenotype may be selected by a variety of methods. Random screening is most common where the mutagenized cells are selected for the ability to produce the desired product or intermediate. Alternatively, selective 20 isolation of mutants can be performed by growing a mutagenized population on selective media where only resistant colonies can develop. Methods of mutant selection are highly developed and well known in the art of industrial microbiology. See, for example, Brock, Supra; DeMancilha et al., Food Chem. 14, 313 (1984). 25 Fermentation media in the present invention comprise sucrose as a carbon substrate. Other carbon substrates such as glucose and fructose may also be present. In addition to the carbon substrate, a suitable fermentation medium contains, for example, suitable minerals, salts, cofactors, buffers and other 30 components, known to those skilled in the art, suitable for the growth of the cultures and promotion of the enzymatic pathway necessary for production of glycerol and its derivatives, for example 1,3-propanediol. 46 Particular attention is given to Co(I1) salts and/or vitamin B 1 2 or precursors thereof in production of 1,3-propanediol. Adenosyl-cobalamin (coenzyme B 12 ) is an important cofactor for dehydratase activity. Synthesis of coenzyme B 12 is found in prokaryotes, 5 some of which are able to synthesize the compound de novo, for example, Escherichia blattae, K/ebsiella species, Citrobacter species, and Clostridium species, while others can perform partial reactions. E. coli, for example, cannot fabricate the corrin ring structure, but is able to catalyze the conversion of cobinamide to corrinoid and can introduce the 10 5'-deoxyadenosyl group. Thus, it is known in the art that a coenzyme B 12 precursor, such as vitamin B 12 , needs be provided in E. coli fermentations. Vitamin B 12 may be added continuously to E. coli fermentations at a constant rate or staged as to coincide with the generation of cell mass, or may be added in single or multiple bolus 15 additions. Although vitamin B 12 is added to the transformed E. co/idescribed herein, it is contemplated that other bacteria, capable of de novo vitamin

B

12 biosynthesis will also be suitable production cells and the addition of vitamin B 12 to these bacteria will be unnecessary. 20 Typically bacterial cells are grown at 25 to 40 0C in an appropriate medium containing sucrose. Examples of suitable growth media for use herein are common commercially prepared media such as Luria Bertani (LB) broth, Sabouraud Dextrose (SD) broth or Yeast medium (YM) broth. Other defined or synthetic growth media may also be used, and the 25 appropriate medium for growth of the particular bacterium will be known by someone skilled in the art of microbiology or fermentation science. The use of agents known to modulate catabolite repression directly or indirectly, e.g., cyclic adenosine 2':3'-monophosphate, may also be incorporated into the reaction media. Similarly, the use of agents known 30 to modulate enzymatic activities (e.g., methyl viologen) that lead to enhancement of 1,3-propanediol production may be used in conjunction 47 with or as an alternative to genetic manipulations with 1,3-propanediol production strains. Suitable pH ranges for the fermentation are between pH 5.0 to pH 9.0, where pH 6.0 to pH 8.0 is typical as the initial condition. 5 Reactions may be performed under aerobic, anoxic, or anaerobic conditions depending on the requirements of the recombinant bacterium. Fed-batch fermentations may be performed with carbon feed, for example, carbon substrate, limited or excess. Batch fermentation is a commonly used method. Classical batch io fermentation is a closed system where the composition of the medium is set at the beginning of the fermentation and is not subject to artificial alterations during the fermentation. Thus, at the beginning of the fermentation, the medium is inoculated with the desired bacterium and fermentation is permitted to occur adding nothing to the system. 15 Typically, however, "batch" fermentation is batch with respect to the addition of carbon source, and attempts are often made at controlling factors such as pH and oxygen concentration. In batch systems, the metabolite and biomass compositions of the system change constantly up to the time the fermentation is stopped. Within batch cultures, cells 20 moderate through a static lag phase to a high growth log phase and finally to a stationary phase where growth rate is diminished or halted. If untreated, cells in the stationary phase will eventually die. Cells in log phase generally are responsible for the bulk of production of end product or intermediate. 25 A variation on the standard batch system is the Fed-Batch system. Fed-Batch fermentation processes are also suitable for use herein and comprise a typical batch system with the exception that the substrate is added in increments as the fermentation progresses. Fed-Batch systems are useful when catabolite repression is apt to inhibit the metabolism of 30 the cells and where it is desirable to have limited amounts of substrate in the media. Measurement of the actual substrate concentration in Fed-Batch systems is difficult and is therefore estimated on the basis of 48 the changes of measurable factors such as pH, dissolved oxygen and the partial pressure of waste gases such as C02. Batch and Fed-Batch fermentations are common and well known in the art and examples may be found in Brock, supra. 5 Continuous fermentation is an open system where a defined fermentation medium is added continuously to a bioreactor and an equal amount of conditioned medium is removed simultaneously for processing. Continuous fermentation generally maintains the cultures at a constant high density where cells are primarily in log phase growth. 10 Continuous fermentation allows for the modulation of one factor or any number of factors that affect cell growth or end product concentration. For example, one method will maintain a limiting nutrient such as the carbon source or nitrogen level at a fixed rate and allow all other parameters to moderate. In other systems, a number of factors affecting 15 growth can be altered continuously while the cell concentration, measured by the turbidity of the medium, is kept constant. Continuous systems strive to maintain steady state growth conditions, and thus the cell loss due to medium being drawn off must be balanced against the cell growth rate in the fermentation. Methods of modulating nutrients and growth 20 factors for continuous fermentation processes as well as techniques for maximizing the rate of product formation are well known in the art of industrial microbiology and a variety of methods are detailed by Brock, supra. It is contemplated that the present invention may be practiced using 25 batch, fed-batch or continuous processes and that any known mode of fermentation would be suitable. Additionally, it is contemplated that cells may be immobilized on a substrate as whole cell catalysts and subjected to fermentation conditions for production of glycerol and glycerol derivatives, such as 1,3-propanediol. 30 In one embodiment, a process for making glycerol, 1,3-propanediol, and/or 3-hydroxypropionic acid from sucrose is provided. The process comprises the steps of culturing a recombinant bacterium, as described 49 above, in the presence of sucrose, and optionally recovering the glycerol, 1,3-propanediol, and/or 3-hydroxypropionic acid produced. The product may be recovered using methods known in the art. For example, solids may be removed from the fermentation medium by centrifugation, filtration, 5 decantation, or the like. Then, the product may be isolated from the fermentation medium, which has been treated to remove solids as described above, using methods such as distillation, liquid-liquid extraction, or membrane-based separation. EXAMPLES 10 The present invention is further defined in the following Examples. It should be understood that these Examples, while indicating preferred embodiments of the invention, are given by way of illustration only. From the above discussion and these Examples, one skilled in the art can ascertain the essential characteristics of this invention, and without 15 departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various uses and conditions. GENERAL METHODS Standard recombinant DNA and molecular cloning techniques 20 described in the Examples are well known in the art and are described by Sambrook, J., Fritsch, E. F. and Maniatis, T. Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, (1989) (Maniatis) and by T. J. Silhavy, M. L. Bennan, and L. W. Enquist, Experiments with Gene Fusions, Cold Spring Harbor Laboratory, 25 Cold Spring Harbor, N.Y. (1984) and by Ausubel, F. M. et al., Current Protocols in Molecular Biology, pub. by Greene Publishing Assoc. and Wiley-Interscience (1987). Materials and methods suitable for the maintenance and growth of bacterial cultures are well known in the art. Techniques suitable for use in 30 the following Examples may be found as set out in Manual of Methods for General Bacteriology (Phillipp Gerhardt, R. G. E. Murray, Ralph N. Costilow, Eugene W. Nester, Willis A. Wood, Noel R. Krieg and G. Briggs 50 Phillips, eds), American Society for Microbiology, Washington, DC. (1994)) or by Thomas D. Brock in Biotechnology: A Textbook of Industrial Microbiology, Second Edition, Sinauer Associates, Inc., Sunderland, MA (1989). All reagents, restriction enzymes and materials described for the 5 growth and maintenance of bacterial cells may be obtained from Aldrich Chemicals (Milwaukee, WI), BD Diagnostic Systems (Sparks, MD), Life Technologies (Rockville, MD), New England Biolabs (Beverly, MA), or Sigma Chemical Company (St. Louis, MO). The meaning of abbreviations is as follows: "s" means second(s), 10 "min" means minute(s), "h" means hour(s), "nm" means nanometers, "pL" means microliter(s), "mL" means milliliter(s), "L" means liter(s), "mM" means millimolar, "M" means molar, "g" means gram(s), "pg" means microgram(s), "bp" means base pair(s), "kbp" means kilobase pair(s), "rpm" means revolutions per minute, "ATCC" means American Type 15 Culture Collection, Manassas, VA, "dH 2 0" means distilled water. EXAMPLE 1 Construction of csc Operon Expression Plasmids This Example illustrates the construction of csc operon expression 20 plasmids pBHRcscBKA and pBHRcscBKAmutB. Genomic DNA was isolated from E. coli strain ATCC1 3281 and digested with EcoRI and BamHI. Fragments approximately 4 kbp in length were isolated by Tris-Borate-EDTA agarose gel electrophoresis and ligated with plasmid vector pLitmus28 (New England Biolabs, Beverly, MA) 25 that had also been digested with EcoRI and BamHI. The resulting plasmids were used to transform E. coli strain DH5alpha (Invitrogen, Carlsbad, CA), and transformants containing the genes required for sucrose utilization were identified by growth on MacConkey sucrose agar (MacConkey agar base from Difco, Sparks, MD) containing 100 g/mL 30 ampicillin. Plasmid DNA was isolated from a colony that had acquired the ability to metabolize sucrose, and the plasmid (designated pScrl; set forth in SEQ ID NO:77) was sequenced to identify the region of DNA necessary 51 for sucrose utilization. The insert was 4140 bp in length and contained putatitve open reading frames homologous to the known E. coli sucrose utilization genes cscB, cscK, and cscA (Jahreis et al., J. Bacteriol. 184:5307-5316, 2002). 5 The csc operon was subsequently moved to plasmid pBHR1 (MobiTec GmbH, Goettingen, Germany) using the following procedure. Plasmid pScrl was digested with Xhol and treated with Klenow fragment to yield blunt ends, followed by digestion with Agel. The resulting 4175 bp fragment containing the csc genes was isolated by gel purification. The 10 plasmid pBHR1 was digested with Agel and Nael, and the resulting 5142 bp fragment was isolated by gel purification. The two gel purified fragments were then ligated, and the resulting plasmid was used to transform E. coli strain DH5alpha. Transformants were selected by growth on Luria Bertani (LB) agar containing 50 g /mL kanamycin. Plasmid DNA 15 was isolated from the transformants, and the sequence of the plasmid was verified. The plasmid was designated pBHRcscBKA (set forth in SEQ ID NO:78). Another expression plasmid was generated by making a single base pair substitution in pBHRcscBKA using the Stratagene QuikChange@ 20 Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA). The thymine base at position 4263 was replaced with guanosine, and the resulting plasmid was designated pBHRcscBKAmutB (set forth in SEQ ID NO:79). This substitution resulted in the replacement of a glutamine residue with histidine in the polypeptide encoded by cscB, a change which was 25 reported to alter the transport capabilities of the homologous protein from E. coli strain EC3132 (Jahreis et al., supra). EXAMPLES 2-4 Construction of Recombinant E. coli Strains Comprisinq the csc Operon 30 These Examples illustrate the construction of recombinant E. coli strains that were transformed with plasmids comprising the csc operon. The consumption of sucrose and the production of the end products 1,3 52 propanediol (PDO) and glycerol from sucrose by these recombinant strains were demonstrated. E. coli strain TTab pSYCO1 09 5 Strain TTab was generated by deletion of the aldB gene from strain TT aldA, described in U.S. Patent No. 7,371,558 (Example 17). Briefly, an aldB deletion was made by first replacing 1.5 kbp of the coding region of aldB in E. coli strain MG1 655 (available from The American Type Culture Collection as ATCC No: 700926) with the FRT-CmR-FRT cassette of the 10 pKD3 plasmid (Datsenko and Wanner, Proc. Nat. Acad. Sci. USA 97:6640-6645, 2000). A replacement cassette was amplified with the primer pair SEQ ID NO:80 and SEQ ID NO:81 using pKD3 as the template. The primer SEQ ID NO:80 contains 80 bp of homology to the 5' end of aldB and 20 bp of homology to pKD3. Primer SEQ ID NO:81 15 contains 80 bp of homology to the 3' end of aldB and 20 bp homology to pKD3. The PCR products were gel-purified and electroporated into MG1 655/pKD46 competent cells (U.S. Patent No. 7,371,558). Recombinant strains were selected on LB plates with 12.5 mg/L of chloroamphenicol. The deletion of the aldB gene was confirmed by PCR, 20 using the primer pair SEQ ID NO:82 and SEQ ID NO:83. The wild-type strain gave a 1.5 kbp PCR product while the recombinant strain gave a characteristic 1.1 kbp PCR product. A P1 lysate was prepared and used to move the mutation to the TT aldA strain to form the TT aldAAaldB::Cm strain. A chloramphenicol-resistant clone was checked by genomic PCR 25 with the primer pair SEQ ID NO:82 and SEQ ID NO:83 to ensure that the mutation was present. The chloramphenicol resistance marker was removed using the FLP recombinase (Datsenko and Wanner, supra) to create strain TTab. Strain TTab was then transformed with pSYCO1 09 (set forth in SEQ ID NO:75), described in U.S. Patent No. 7,371,558, to 30 generate strain TTab pSYCO1 09. As described in the cited references, strain TTab is a derivative of E. coli strain FM5 (ATCC No. 53911) containing the following 53 modifications: deletion of gpK, gidA, ptsHl, crr, edd, arcA, mgsA, qor, ackA, pta, aldA and aldB genes; upregulation of galP, glk, btuR, ppc, and yqhD genes; and 5 downregulation of gapA gene. Plasmid pSYCO1 09 contains genes encoding a glycerol production pathway (DAR1 and GPP2) and genes encoding a glycerol dehydratase and associated reactivating factor (dhaB123, dhaX, orfX, orfY). Strain TTab/pSYCO1 09 was transformed with each of the two csc 10 operon overexpression plasmids pBHRcscBKA and pBHRcscBKAmutB, described in Example 1. Transformants were selected by growth on LB agar containing 50 g/mL of spectinomycin and 50 g/mL of kanamycin. Individual colonies were picked and grown overnight at 34 0C with shaking (250 rpm) in LB broth with the same antibiotics. The control strain 15 TTab/pSYCO1 09 was grown under identical conditions with the exception of the kanamycin. These overnight cultures were diluted into TM3 medium containing 10.5 g/L sucrose to an optical density of 0.01 units measured at 550 nm. TM3 is a minimal medium containing 13.6 g/L KH 2

PO

4 , 2.04 g/L citric acid 20 dihydrate, 2 g/L magnesium sulfate heptahydrate, 0.33 g/L ferric ammonium citrate, 0.5 g/L yeast extract, 3 g/L ammonium sulfate, 0.2 g/L CaCl 2 -2H 2 0, 0.03 g MnSO 4

-H

2 0, 0.01 g/L NaCI, 1 mg/L FeSO 4 -7H 2 0, 1 mg/L, CoCl 2 -6H 2 0, 1 mg/L ZnSO 4 -7H 2 0, 0.1 mg/L CuSO 4 -5H 2 0, 0.1 mg/L

H

3 B0 4 , 0.1 mg/L NaMoO 4 -2H 2 0 and sufficient NH 4 0H to provide a final 25 pH of 6.8. Vitamin B 12 was added to the medium to a concentration of 0.1 mg/L. The cultures were incubated at 34 0C with shaking (225 rpm) for 24 hours. Aliquots were removed at 0, 5, 8, 11, 14, 17, 20 and 23 hours after inoculation, and the concentrations of sucrose, glycerol and 1,3 propanediol (PDO) in the broth were determined by high performance 30 liquid chromatography. Chromatographic separation was achieved using an Aminex HPX 87P column (Bio-Rad, Hercules, CA) with an isocratic mobile phase of 54 dH 2 0 at a flow rate of 0.5 mL/min and a column temperature of 60 C. Eluted compounds were quantified by refractive index detection with reference to a standard curve prepared from commercially purchased pure compounds dissolved to known concentrations in the TM3 medium. 5 Retention times were sucrose at 12.2 min, 1,3-propanediol at 17.9 min, and glycerol at 23.6 min. Both csc expression plasmids (Examples 3 and 4) resulted in metabolism of sucrose and production of PDO and glycerol while the parent control strain (Example 2, Comparative) was unable to metabolize io sucrose or produce PDO or glycerol under these conditions (see Tables 2 4). The data points given in the tables represents the average of measurements made on two duplicate cultures. Table 2 15 Sucrose consumption Sucrose (g/L) Example 2, Time Comparative Example 3 Example 4 (h) Control Strain +pBHRcscBKA +pBHRcscBKAmutB 0 10.48 10.48 10.48 6 10.14 10.05 10.08 12 10.34 9.87 10.17 18 10.28 7.31 10.17 24 10.32 0.65 10.13 30 10.37 0.00 8.44 36 10.36 0.00 3.32 42 10.33 0.00 0.00 55 Table 3 PDO Production PDO (g/L) Example 2, Time Comparative Example 3 Example 4 (h) Control Strain +pBHRcscBKA +pBHRcscBKAmutB 0 0.00 0.00 0.00 6 0.00 0.00 0.00 12 0.00 0.00 0.00 18 0.00 0.41 0.00 24 0.00 2.20 0.02 30 0.00 3.15 0.24 36 0.00 3.15 1.35 42 0.00 3.06 2.82 5 Table 4 Glycerol production Glycerol (g/L) Example 2, Time Comparative Example 3 Example 4 (h) Control Strain +pBHRcscBKA +pBHRcscBKAmutB 0 0.00 0.00 0.00 6 0.00 0.00 0.00 12 0.00 0.08 0.00 18 0.00 0.96 0.00 24 0.00 3.24 0.00 30 0.00 2.54 0.60 36 0.00 2.52 2.33 42 0.00 2.49 2.98 Where the terms "comprise", "comprises", "comprised" or "comprising" are used in this specification, they are to be interpreted as 10 specifying the presence of the stated features, integers, steps or components referred to, but not to preclude the presence or addition of one or more other feature, integer, step, component or group thereof. Further, any prior art reference or statement provided in the specification is not to be taken as an admission that such art constitutes, 15 or is to be understood as constituting, part of the common general knowledge in Australia. 56

Claims (18)

1. A recombinant bacterium comprising in its genome or on at least one recombinant construct: 5 (a) one or more nucleotide sequences encoding a polypeptide or a polypeptide complex having sucrose transporter activity; (b) a nucleotide sequence encoding a polypeptide having fructokinase activity; (c) a nucleotide sequence encoding a polypeptide having 10 sucrose hydrolase activity; and (d) one or more nucleotide sequences encoding a polypeptide or a polypeptide complex having dehydratase activity; wherein (a), (b), (c) and (d) are each operably linked to the same or a different promoter, and wherein at least one of (a), (b), (c) or (d): (i) is on 15 a recombinant construct; (ii) encodes a polypeptide comprising a non native polypeptide; (iii) comprises a foreign gene; or (iv) is any combination of (i), (ii) or (iii), further wherein said recombinant bacterium has improved ability to metabolize sucrose to produce a product selected from the group 20 consisting of glycerol, 1,3-propanediol and 3-hydroxypropionic acid compared to an equivalent microorganism lacking (a), (b), (c) and (d).

2. The recombinant bacterium of claim 1, wherein the polypeptide or polypeptide complex having dehydratase activity is selected from the 25 group consisting of: (a) a polypeptide or polypeptide complex having glycerol dehydratase activity classified as EC 4.2.1.30; and (b) a polypeptide or polypeptide complex having diol dehydratase activity classified as EC 4.2.1.28. 30

3. The recombinant bacterium of claim 2, wherein the polypeptide complex having glycerol dehydratase activity comprises: 57 (a) a first subunit having at least 95% sequence identity, based on a Clustal V method of alignment, when compared to an amino acid sequence as set forth in SEQ ID NO:1 2; (b) a second subunit having at least 95% sequence identity, 5 based on a Clustal V method of alignment, when compared to an amino acid sequence as set forth in SEQ ID NO:14; and (c) a third subunit having at least 95% sequence identity, based on a Clustal V method of alignment, when compared to an 10 amino acid sequence as set forth in SEQ ID NO:1 6.

4. The recombinant bacterium of any one of claims 1 to 3, comprising: (a) a nucleotide sequence encoding a polypeptide having glycerol-3-phosphate dehydrogenase activity; 15 (b) a nucleotide sequence encoding a polypeptide having glycerol-3-phosphatase activity; or (c) both (a) and (b).

5. The recombinant bacterium of claim 4, wherein said polypeptide 20 having glycerol-3-phosphate dehydrogenase activity has at least 95% identity, based on the Clustal V method of alignment, when compared to an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 and 4. 25

6. The recombinant bacterium of claim 4, wherein said polypeptide having glycerol-3-phosphatase activity has at least 95% identity, based on the Clustal V method of alignment, when compared to an amino acid sequence selected from the group consisting of SEQ ID NOs: 6 and 8. 30

7. The recombinant bacterium of any one of claims 1 to 6, wherein the polypeptide having sucrose transporter activity has at least 95% sequence identity, based on a Clustal V method of alignment, when compared to an 58 amino acid sequence as set forth in SEQ ID NO:24, SEQ ID NO:26, or SEQ ID NO:28.

8. The recombinant bacterium of any one of claims 1 to 6, wherein the 5 polypeptide complex having sucrose transporter activity comprises: (a) a first subunit having at least 95% sequence identity, based on a Clustal V method of alignment, when compared to an amino acid sequence as set forth in SEQ ID NO:30; (b) a second subunit having at least 95% sequence identity, 10 based on a Clustal V method of alignment, when compared to an amino acid sequence as set forth in SEQ ID NO:32; and (c) a third subunit having at least 95% sequence identity, based on a Clustal V method of alignment, when compared to an 15 amino acid sequence as set forth in SEQ ID NO:34.

9. The recombinant bacterium of any one of claims 1 to 6, wherein the polypeptide complex having sucrose transporter activity comprises: (a) a first subunit having at least 95% sequence identity, based 20 on a Clustal V method of alignment, when compared to an amino acid sequence as set forth in SEQ ID NO:36; (b) a second subunit having at least 95% sequence identity, based on a Clustal V method of alignment, when compared to an amino acid sequence as set forth in SEQ ID NO:38; 25 (c) a third subunit having at least 95% sequence identity, based on a Clustal V method of alignment, when compared to an amino acid sequence as set forth in SEQ ID NO:40; and (d) a fourth subunit having at least 95% sequence identity, based on a Clustal V method of alignment, when compared 30 to an amino acid sequence as set forth in SEQ ID NO:42.

10. The recombinant bacterium of any one of claims 1 to 9, wherein the 59 polypeptide having fructokinase activity has at least 95% sequence identity, based on a Clustal V method of alignment, when compared to an amino acid sequence as set forth in SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:85, 5 or SEQ ID NO:87.

11. The recombinant bacterium of any one of claims 1 to 10, wherein the polypeptide having sucrose hydrolase activity has at least 95% sequence identity, based on a Clustal V method of alignment, when io compared to an amino acid sequence as set forth in SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, or SEQ ID NO:68.

12. The recombinant bacterium of any one of claims 1 to 6, wherein the 15 polypeptide having sucrose transporter activity consists of the sequence set forth in SEQ ID NO:26.

13. The recombinant bacterium of any one of claims 1 to 9, wherein the polypeptide having fructokinase activity consists of the sequence set forth 20 in SEQ ID NO:48.

14. The recombinant bacterium of any one of claims 1 to 10, wherein the polypeptide having sucrose hydrolase activity consists of the sequence set forth in SEQ ID NO:58. 25

15. The recombinant bacterium of any one of claims 1 to 14 wherein said bacterium is selected from the group consisting of the genera: Escherichia, Klebsiella, Citrobacter, and Aerobacter. 30

16. The recombinant bacterium of claim 15 wherein said bacterium is Escherichia coli. 60

17. A process for making glycerol, 1,3-propanediol and/or 3 hydroxypropionic acid from sucrose comprising: (a) culturing the recombinant bacterium of any one of claims 1 to 14 in the presence of sucrose; and 5 (b) optionally, recovering the glycerol, 1,3-propanediol and/or 3 hydroxypropionic acid produced.

18. A process for making glycerol, 1,3-propanediol and/or 3 hydroxypropionic acid from sucrose comprising: 10 (a) culturing the recombinant bacterium of claim 15 in the presence of sucrose; and (b) optionally, recovering the glycerol, 1,3-propanediol and/or 3 hydroxypropionic acid produced. 15 61 2014274643 12 Dec 2014 2014274643 12 Dec 2014 2014274643 12 Dec 2014 2014274643 12 Dec 2014 2014274643 12 Dec 2014 2014274643 12 Dec 2014 2014274643 12 Dec 2014 2014274643 12 Dec 2014 2014274643 12 Dec 2014 2014274643 12 Dec 2014 2014274643 12 Dec 2014 2014274643 12 Dec 2014 2014274643 12 Dec 2014 2014274643 12 Dec 2014 2014274643 12 Dec 2014 2014274643 12 Dec 2014 2014274643 12 Dec 2014 2014274643 12 Dec 2014 2014274643 12 Dec 2014 2014274643 12 Dec 2014 2014274643 12 Dec 2014 2014274643 12 Dec 2014 2014274643 12 Dec 2014 2014274643 12 Dec 2014 2014274643 12 Dec 2014 2014274643 12 Dec 2014 2014274643 12 Dec 2014 2014274643 12 Dec 2014 2014274643 12 Dec 2014 2014274643 12 Dec 2014 2014274643 12 Dec 2014 2014274643 12 Dec 2014 2014274643 12 Dec 2014 2014274643 12 Dec 2014 2014274643 12 Dec 2014 2014274643 12 Dec 2014 2014274643 12 Dec 2014 2014274643 12 Dec 2014 2014274643 12 Dec 2014 2014274643 12 Dec 2014 2014274643 12 Dec 2014 2014274643 12 Dec 2014 2014274643 12 Dec 2014 2014274643 12 Dec 2014 2014274643 12 Dec 2014 2014274643 12 Dec 2014 2014274643 12 Dec 2014 2014274643 12 Dec 2014 2014274643 12 Dec 2014 2014274643 12 Dec 2014 2014274643 12 Dec 2014 2014274643 12 Dec 2014 2014274643 12 Dec 2014 2014274643 12 Dec 2014 2014274643 12 Dec 2014 2014274643 12 Dec 2014 2014274643 12 Dec 2014 2014274643 12 Dec 2014 2014274643 12 Dec 2014 2014274643 12 Dec 2014 2014274643 12 Dec 2014 2014274643 12 Dec 2014 2014274643 12 Dec 2014 2014274643 12 Dec 2014 2014274643 12 Dec 2014 2014274643 12 Dec 2014 2014274643 12 Dec 2014 2014274643 12 Dec 2014 2014274643 12 Dec 2014 2014274643 12 Dec 2014 2014274643 12 Dec 2014 2014274643 12 Dec 2014 2014274643 12 Dec 2014 2014274643 12 Dec 2014 2014274643 12 Dec 2014 2014274643 12 Dec 2014 2014274643 12 Dec 2014 2014274643 12 Dec 2014 2014274643 12 Dec 2014 2014274643 12 Dec 2014 2014274643 12 Dec 2014 2014274643 12 Dec 2014 2014274643 12 Dec 2014 2014274643 12 Dec 2014 2014274643 12 Dec 2014 2014274643 12 Dec 2014 2014274643 12 Dec 2014 2014274643 12 Dec 2014 2014274643 12 Dec 2014 2014274643 12 Dec 2014 2014274643 12 Dec 2014 2014274643 12 Dec 2014 2014274643 12 Dec 2014 2014274643 12 Dec 2014 2014274643 12 Dec 2014 2014274643 12 Dec 2014 2014274643 12 Dec 2014 2014274643 12 Dec 2014 2014274643 12 Dec 2014 2014274643 12 Dec 2014 2014274643 12 Dec 2014 2014274643 12 Dec 2014 2014274643 12 Dec 2014 2014274643 12 Dec 2014 2014274643 12 Dec 2014 2014274643 12 Dec 2014 2014274643 12 Dec 2014 2014274643 12 Dec 2014 2014274643 12 Dec 2014 2014274643 12 Dec 2014 2014274643 12 Dec 2014 2014274643 12 Dec 2014 2014274643 12 Dec 2014 2014274643 12 Dec 2014 2014274643 12 Dec 2014 2014274643 12 Dec 2014 2014274643 12 Dec 2014 2014274643 12 Dec 2014 2014274643 12 Dec 2014 2014274643 12 Dec 2014 2014274643 12 Dec 2014 2014274643 12 Dec 2014 2014274643 12 Dec 2014 2014274643 12 Dec 2014 2014274643 12 Dec 2014 2014274643 12 Dec 2014 2014274643 12 Dec 2014 2014274643 12 Dec 2014 2014274643 12 Dec 2014 2014274643 12 Dec 2014 2014274643 12 Dec 2014 2014274643 12 Dec 2014 2014274643 12 Dec 2014 2014274643 12 Dec 2014 2014274643 12 Dec 2014 2014274643 12 Dec 2014 2014274643 12 Dec 2014 2014274643 12 Dec 2014 2014274643 12 Dec 2014 2014274643 12 Dec 2014 2014274643 12 Dec 2014